Settings for recombinant adenoviral-based vaccines

ABSTRACT

Described are new uses of recombinant adenoviral vectors in vaccination regimens, such as prime/boost set-ups and subsequent vaccinations and applications for gene therapy. Moreover, also described are new assays to determine the best regimen for applying the most suitable recombinant viral vector in a vaccination or gene therapy setting.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of co-pending U.S. patent applicationSer. No. 12/583,628, filed Aug. 24, 2009, which application is adivisional of U.S. Ser. No. 11/105,725, filed Apr. 14, 2005, now U.S.Pat. No. 7,598,078, issued Oct. 6, 2009, which application is acontinuation of International Patent Appln. No. PCT/EP03/050748, filedon Oct. 23, 2003, designating the United States of America, andpublished, in English, as PCT International Publication No. WO2004/037294 A2 on May 6, 2004, which claims priority under 35 U.S.C.§119 to International Patent Application No. PCT NL02/00671, filed Oct.23, 2002, the contents of the entirety of each of which are incorporatedby this reference.

TECHNICAL FIELD

The invention relates generally to the field of biotechnology, and moreparticularly to the field of medicine, in particular to the field ofvaccination using recombinant adenoviral vectors. The disclosurespecifically relates to the production and controlled use of vaccinesbased on adenoviruses derived from different serotypes.

BACKGROUND

Many different kinds of vaccines are being employed to preventpathogenic entities to enter the body or to prevent the pathogenicentities to spread and cause illnesses. Vaccines that are being appliednowadays and/or vaccines that are being tested in different stages ofdevelopment include whole-inactivated viruses, (live-) attenuatedviruses, peptide vaccines, (naked) DNA vaccines, sub-unit vaccines andvaccines that are based on (relatively) harmless viruses that harbor anantigenic determinant from the pathogenic entity towards which thevaccine is directed. Examples of such “vaccine carriers” are influenzavirus, alphaviruses such as Semliki Forest Virus or Sindbis virus, andadenoviruses. Wild-type adenoviruses are known to cause relatively milddiseases such as common colds. To date, over 50 different adenovirusserotypes have been identified, subdivided into six subgroups based ontheir sequence homologies and hemagglutination abilities. Recombinantadenoviruses are being extensively tested in HIV vaccine clinical trialsand in vaccines against malaria (WO 01/02607; WO 02/22080; WO 01/21201;Sullivan et al. 2000; Shiver et al. 2002). The results that wereobtained in these studies clearly show that adenoviruses provide anexcellent tool for delivery of the antigen to the host. One couldenvision an endless list of other pathogens that could be targeted byusing the adenovirus as an antigen carrier providing proper protection.Such pathogens include, but are not limited to, viruses, bacteria,yeasts, fungi, etc.

However, a few important drawbacks exist when the most common andprobably the best-studied adenovirus serotype, Adenovirus 5 (Ad5) isused. As has been described extensively elsewhere (PCT InternationalPublication WO 00/70071), it is known that most people across the worldhave encountered an Ad5 infection at least once in their lifetimes. Thisexposure results in a level of neutralizing antibodies that isrelatively high and causes a rapid clearance from the system. Moreover,it is known that almost all Ad5-derived recombinant vectors end up inthe liver. This phenomenon presumably prevents the recombinant vector(based on Ad5) from very efficiently entering the antigen-presentingcells such as dendritic cells. The art has recognized that there was aneed for alternative adenoviruses that would not home to the liver, butrather would be targeted to the cells involved in the immune system. Oneway of triggering this was by employing the receptor- or cell-bindingmoiety of the adenovirus. This moiety was swapped from certainadenoviruses not having a tropism for liver cells to Ad5. An example ofsuch a recombinant adenovirus is Ad5fib16, which is a recombinantadenovirus based on Ad5, but carrying the tropism-determining part ofthe fiber of adenovirus serotype 16 in its capsid (see, PCTInternational Publications WO 00/03029 and WO 02/24730).

SUMMARY OF THE INVENTION

Disclosed are methods and means for vaccination purposes usingrecombinant adenoviral vectors. Provided is a use of a recombinantadenovirus vector of a first serotype for the preparation of amedicament for the treatment or prevention of a disease in a human oranimal treated with a recombinant adenovirus vector of a secondserotype, wherein the first serotype is different from the secondserotype. Also provides is the use of a recombinant adenovirus vector ofa first serotype for the preparation of a medicament for the treatmentor prevention of a disease in a human or animal having an antibody titeragainst an adenovirus of a second serotype, wherein the first serotypeis different from the second serotype. Furthermore provided is a kit ofparts comprising a priming composition and a boosting composition, bothcompositions comprising: a recombinant adenovirus vector; a heterologousnucleic acid of interest present in the vector; and a pharmaceuticallyacceptable carrier, wherein the recombinant adenovirus vector of thepriming composition is from a different serotype than the recombinantadenovirus vector of the boosting composition. Also provided is a methodfor determining the titer of neutralizing antibodies in a blood sample,wherein the neutralizing antibodies are directed against a virus,comprising the steps of: obtaining a sample; culturing host cells;infecting the host cells with recombinant viral vectors comprising atransgene, in the presence of the sample; and determining the activityof a protein encoded by the transgene. Further provided is a method fordetermining the titer of neutralizing antibodies in a blood sample,wherein the neutralizing antibodies are directed against a virus,comprising the steps of: obtaining a sample; culturing host cells;infecting the host cells with recombinant viral vectors in the presenceof the sample; and determining the number of viral genomes per cell.

Provided are methods and means that solve problems in the field ofvaccination. Provided is the use of a recombinant adenovirus vector of afirst serotype for the preparation of a medicament for the treatment orprevention of a disease in a human or animal treated with a recombinantadenovirus vector of a second serotype, wherein the first serotype isdifferent from the second serotype. The disclosure also relates to theuse of a recombinant adenovirus vector of a first serotype for thepreparation of a medicament for the treatment or prevention of a diseasein a human or animal having an antibody titer against an adenovirus of asecond serotype, wherein the first serotype is different from the secondserotype. In certain embodiments, the second serotype is selected fromthe group consisting of: Ad11, Ad26, Ad34, Ad35, Ad46 and Ad49, andwherein the first serotype is selected from the group consisting of:Ad11, Ad26, Ad34, Ad35, Ad46 and Ad49. In certain embodiments, the firstserotype is comprised in a vaccine composition (normally a boostcomposition), while the second serotype is part of a primingcomposition. It is to be understood that it is part of the disclosurethat if an individual does not have a high titer of neutralizingantibodies against an adenovirus serotype that is known in the art, suchas Ad5, Ad2, Ad3, Ad4, Ad7 and Ad12, that the priming composition maycomprise a vaccine based on such known adenovirus serotype, preferablyAd5, while the following composition (boost) should comprise anotheradenovirus serotype for which the individual also does not havesignificantly high levels of neutralizing antibodies in its serum. Ofcourse, such following compositions may comprise an adenovirus vectorselected from the same groups, as long as the first and second serotypesare different. If the subject has a significantly high titer to a secondadenovirus (obtained through a general infection, or through activevaccination, or through a gene therapy application) the vector of choicefor the first adenovirus serotype should be different from the secondadenovirus serotype. “Significantly high” in this context means thatsuch titers hamper the immune response elicited by the vector beingapplied, due to neutralization of the vector, hence, leading to thechoice of a serotype that would not encounter titers of neutralizingantibodies that cause the immune response to be so low that a protectiveeffect of the vaccine is not accomplished. Moreover, it is also to beunderstood that if a vaccine regimen requires more than two shots(prime/boost), but rather extra subsequent shots (prime/boost/boost,etc.), that this is also part of the invention: the subsequent boostcompositions should always (if they comprise an adenovirus vector)comprise an adenovirus vector that is different from the adenovirusvectors that have been used previously, at least as long the titers ofneutralizing antibodies hamper the immune response required.

“Based on” or “derived from” as used herein means that a gene deliveryvehicle, such as a recombinant adenovirus vector, originates from acertain wild-type adenovirus serotype as they have been recognized inthe art. This means in general that certain parts of the genome aredeleted to prevent replication (such as a deletion of the E1 region),but it also means that other mutations, deletions, naturally occurringchimeras, additions of nucleic acid, etc., may or may not be present inthe recombinant adenoviral vector, as long as the capsid proteinstowards which the neutralizing antibodies present in the serum frominfected or vaccinated individuals are sufficiently different from onecomposition to the other. For example, if the backbone of therecombinant vector (this means generally all elements except theimmunogenic and tropism-determining parts of the capsid) is identicalbetween prime and boost compositions, this is still considered part ofthe invention, since the immune response towards such vectors having thesame or similar backbone is still different.

The recombinant adenovirus vectors of the first and second serotypes maycomprise essentially the same heterologous nucleic acid of interest. Forvaccination purposes, it is generally required that the same antigen, orthe nucleic acid encoding that antigen, is administered several times.“Essentially” as used herein refers to the idea that the antigen mightbe slightly different, but should still elicit an immune response thatwould fully (or at least sufficiently) protect the vaccinated individualfrom the pathogen. Generally, recombinant adenoviruses harbor thenucleic acid encoding the heterologous protein in the E1 region that isnormally deleted from the genome.

The heterologous nucleic acid may encode a viral antigen. Morepreferably, the viral antigen is an Ebola virus antigen, a measles virusantigen, or a West Nile virus antigen. Such antigens can be obtained bysequencing the genomes of the wild-type strains of the differentviruses, subcloning the nucleic acids encoding the antigenicdeterminants from such genomes, and cloning them into the adenoviralgenomic sequence.

The viral antigen may be an antigen from a retrovirus such as HumanImmunodeficiency Virus (HIV), a Simian Immunodeficiency Virus (SIV), andantigens derived from Feline Immunodeficiency Virus (FIV). The HIV, SIVor FIV antigen may be gag, env, nef, pol and/or combinations thereof.

The heterologous nucleic acid present in the first and second serotypemay encode a malaria antigen, such as the circumsporozoite (CS) or LSA-1antigen from Plasmodium yoelii or Plasmodium falciparum, or functionalequivalents or antigenic determinants/parts or derivatives thereof.

Further provided is a kit of parts comprising a priming composition anda boosting composition, both compositions comprising: a recombinantadenovirus vector; a heterologous nucleic acid of interest present inthe vector; and a pharmaceutically acceptable carrier, wherein therecombinant adenovirus vector of the priming composition is from adifferent serotype than the recombinant adenovirus vector of theboosting composition. The recombinant adenovirus vector of the primingcomposition may be of a serotype selected from the group consisting ofAd11, Ad26, Ad34, Ad35, Ad46, and Ad49. Also preferred is a kit of partsaccording to the invention, wherein the recombinant adenovirus vector ofthe boosting composition is of a serotype selected from the groupconsisting of Ad11, Ad26, Ad34, Ad35, Ad46, and Ad49. It is still to beunderstood that other adenovirus serotypes may be comprised in the kitof parts hereof as long as the individual that is to be treated does notcarry neutralizing antibodies to significantly high titers against thatparticular adenovirus serotype and as long as the second and firstserotypes are different.

In one embodiment, also provided is the use of a recombinant adenovirusvector derived from Ad11 for the preparation of a medicament in thetreatment of a human or animal suffering from, or at risk of, a diseasecaused by a virus. Besides Ad35, Ad11 is a highly preferred serotypesince most people in the world do not carry neutralizing antibodiesagainst Ad11.

Also provided is a method for determining the titer of neutralizingantibodies in a human- or animal-derived blood sample, wherein theneutralizing antibodies are directed against a virus, comprising thesteps of: obtaining a sample; culturing host cells; infecting the hostcells with recombinant viral vectors comprising a transgene, in thepresence of the sample; and determining the activity of a proteinencoded by the transgene. Preferably, the determined activity iscompared to a standard value. Even more preferred are methods whereinthe transgene encodes a protein selected from the group consisting of:luciferase, Green Fluorescent Protein (GFP) and LacZ. Also provided is amethod for determining the titer of neutralizing antibodies in a bloodsample, wherein the neutralizing antibodies are directed against avirus, comprising the steps of: obtaining a sample; culturing hostcells; infecting the host cells with recombinant viral vectors in thepresence of the sample; and determining the number of viral genomes percell. The number of viral genomes may be compared to a standard value.Also preferred are methods, wherein the number of viral genomes per cellis determined by Quantitative-PCR (Q-PCR).

In a certain embodiment, the methods are applied for determining thetiter of neutralizing antibodies that are directed against anadenovirus. These antibodies might have been raised during previousvaccinations, prime and/or boost injections or through natural occurringinfections. For determining the titer of neutralizing antibodies againstan adenovirus, it is preferred to use a recombinant adenoviral vectorherein. The host cells used in a method hereof should be receptive forviral infection, preferably for adenoviral infection. A preferred cellline is the A549 cell line. Since titers may be very high, it is usefulto make a curve of serial dilutions of the sample and to compare thiswith a standard curve.

It is very useful to know what titers of neutralizing antibodies arepresent in the serum of the individual to be treated. The methods knownin the art are not considered accurate and useful for high throughputuse. The method provided herein ensures a way of determining thepresence of neutralizing antibodies against all different adenovirusserotypes known in the art. This can then be followed by a regimen asprovided by the invention in which adenovirus vectors based on differentserotypes are used in subsequent vaccine applications, such asprime/boosts. It is to be understood that the method is not limited tothe transgenes as described in the present disclosure, or to thematerials such as antibodies as described in the provided example. Themethod can be executed by using a kit of parts comprising a plate, astandard curve of diluted antibodies for possibly all serotypes knownand possibly materials such as buffers and antisera for detection.

This disclosure relates to methods and means to overcome at least partof the limitations of adenovirus-based vaccines. It has been recognizedin the art that a series of vaccine applications would render a betterand more potent immune response towards a certain immunogenic antigen.In the HIV vaccine studies (WO 01/02607; WO 02/22080), several regimenswere tested, including the use of naked DNA encoding the antigen, as apriming composition, after which a boosting composition comprising arecombinant Ad5 vector was applied. Similar regimens were followed inobtaining a specific response against malaria antigens in other studies(WO 01/21201). It has been suggested by some to use different (lowneutralized) serotypes of adenovirus in different rounds of vaccinationand gene therapy applications (Parks et al. 1999; Mack et al. 1997; Hsuet al. 1992; Moffat et al. 2000; Kass-Eisler et al. 1996; Mastrangeli etal. 1996; Roy et al. 1998; Lubeck et al. 1997). However, the inventionrealizes that such regimens are feasible for subsequent series ofvaccinations, applying different antigens directed towards differentpathogens, but using the same serotype in one prime/boost setting wouldstill render the boost immune response weaker if the same serotype wouldhave been used in the priming composition. Settings in which differentserotypes are used in a prime/boost set-up for the same vaccine have notbeen suggested, nor have they been used in the art. The art describeseither the use of the same serotype (mostly Ad5) in prime/boost set-upsor the use of different kinds of compositions like, for instance, nakedDNA encoding the antigen, and a certain serotype (being mostly Ad5) ascarrier of the DNA encoding the antigen in prime/boost settings. We nowshow for the first time that pre-existing immunity against a well-knownand widely used vector as Ad5 can be overcome by using a recombinantadenoviral vector that is based on an adenovirus serotype that has a lowprevalence in humans and that is not neutralized by antibodies in alarge percentage of the worldwide population.

Also provided are methods and means for repeated vaccinationapplications, using different serotypes from the same subgroup.Moreover, we disclose that, indeed, subjects who are immunized withAd5-based vectors do not raise antibodies that are directed against asubsequent adenovirus serotype such as Ad35 or Ad11, while the titer ofantibodies directed against the antigen (measles antigen H, or SIV-gag)is higher when an Ad5-Ad35 regimen is applied as compared to an Ad5-Ad5regimen. This result strongly indicates that subsequent applications ofan adenovirus of the same subgroup are not very efficient invaccination, while subsequent applications of adenoviruses of differentserotype are. These results also strongly suggest that an individualthat has encountered an Ad5 infection in the past should preferablyreceive a priming vaccine composition comprising an adenovirus that isat least different from Ad5, while the boosting composition (ifapplicable) should also comprise yet another serotype that has neverinfected that particular individual.

Also disclosed is that cross-neutralization is not an important issue.It was widely believed that a certain extent of cross-neutralizationcould or may prevent the use of different adenoviruses that areextremely similar. As disclosed herein, sera that harbor neutralizingantibodies against Ad35 do not, in most cases, contain neutralizingantibodies against Ad11 and vice versa. The disclosure, therefore, makesit now possible to use prime/boost vaccination applications in which thepriming composition comprises one adenovirus serotype, while theboosting composition comprises an adenovirus from another serotype. Theinvention discloses which adenovirus serotypes are suitable for suchsettings. Preferred serotypes that are used in prime/boost applicationsaccording to the invention are the subgroup B serotypes Ad11 and Ad35,since these serotypes encounter neutralizing antibodies in only a verylimited number of human sera, while humans that have encountered Ad11 intheir lifetime most likely do not contain neutralizing antibodiesagainst Ad35, and vice versa. The chance of encountering both serotypesin one lifetime seems to be extremely slim. The use of such adenovirusserotypes, of course, would render a vaccine that needs priming andboosting compositions for a proper immune response more potent than avaccine that is built up from serotypes that are likely to encounterneutralizing antibodies, such as Ad5.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph showing the percentage of serum samples positivefor neutralization (IC90 values) for Ad5, −35 and −11 in six differentlocations (Belgium: BE; United Kingdom: UK; United States: “US1” and“US2,” Japan and The Netherlands).

FIG. 2 is a graph showing the worldwide average percentage of serumsamples positive for neutralization (IC90 values) for Ad5, −35 and −11.

FIG. 3 is a bar graph showing the percentage sera samples that showneutralizing activity to Ad5-Ad11, Ad5-Ad35, Ad11-Ad35 andAd11-Ad35-Ad5.

FIG. 4 is a map of pAdapt1MV-H.

FIG. 5 is a map of pAdapt5.SIVgag.

FIG. 6 is a map of pAdapt35MV-H.

FIG. 7 is a map of pAdapt535.SIVgag.

FIG. 8 shows the immunization schedule for the mice study.

FIG. 9 shows the anti-measles IgG response in mice after Ad5.MV-H (left)and Ad35.MV-H (right) vaccination as measured by ELISA assay.

FIG. 10 shows the anti-measles T-cell response in mice after Ad5.MV-Hand Ad35.MV-H vaccination as measured by ELISPOT assay. Left: a bargraph showing the values of MV-H-specific cells per spleen. Right: a bargraph showing the percentage values of MV-H-specific T-cells.

FIG. 11 shows the anti-measles antibody response in cynomolgus monkeysafter Ad5.MV-H and Ad35.MV-H vaccination as measured by MV-H-specificimmunofluorescence assay.

FIG. 12 is a table showing immunization schedule and time points forblood sampling for the in vivo study in cynomolgus monkeys.

FIG. 13 shows the anti-measles antibody response in cynomolgus monkeysafter Ad5.MV-H prime and Ad5.MV-H or Ad35.MV-H boost vaccination asmeasured by MV-H-specific immunofluorescence assay.

FIG. 14 depicts SIV-gag-specific cellular and humoral responses in naïvemice using Ad5-SIVgag and Ad35-SIVgag-recombinant viruses.

FIG. 15 shows the mapping of D^(b)-restricted and K^(b)-restrictedT-lymphocyte epitopes within SW-gag using C57/BL6 mice. P78 is SEQ IDNO:1; AM7 is SEQ ID NO:2; AQ9 is SEQ ID NO:3; AL11 is SEQ ID NO:4; P19is SEQ ID NO:5; KV9 is SEQ ID NO:6; KI10 is SEQ ID NO:7; LV8 is SEQ IDNO:8; LV10 is SEQ ID NO:9.

FIG. 16 shows Ad5-specific and Ad35-specific Nab titers in humans.

FIG. 17 depicts humoral and cellular immune responses to Ad5-SIVgag andAd35-SIVgag in mice pre-immunized with one shot of Ad5-Empty.

FIG. 18 depicts humoral and cellular immune responses to Ad5-SIVgag andAd35-SIVgag in mice pre-immunized with two shots of Ad5-Empty.

FIG. 19 shows immune responses upon several heterologous prime/boostregimens in naïve mice.

FIG. 20 shows immune responses upon several heterologous prime/boostregimens in Ad5-Empty pre-immunized mice.

FIG. 21 is a map of pPCRScript-Ad11.

FIG. 22 is a map of pAdApt11.

FIG. 23 is a map of pAdApt11.dH.

FIG. 24 is a map of pAdApt535.Luc.

FIG. 25 is a map of pAdApt535.

FIG. 26 is a map of pAdApt535.overlap.dPr11.

FIG. 27 is a map of pAdApt511.dH.

FIG. 28 is a map of pAdApt511.

FIG. 29 is a map of pWE.Ad11.dNdeI.

FIG. 30 is a map of pWE.Ad11.dNdeI.dPr.

FIG. 31 is a map of pWE.Ad11.dNdeI.dH.dPr.

FIG. 32 is a comparison between an ARIA and an ATEIA assay.

FIG. 33 shows different transgene activities using diluted numbers ofcells per well in a titration assay.

FIGS. 34A through 34D show the different transgene activities andgenomes per cell.

FIG. 35 shows the neutralization capacity of Ad5-positive and -negativeserum +/− spiking of IgG isolated from Ad5-positive serum.

DETAILED DESCRIPTION OF THE INVENTION

Since it was found that many individuals in different populations carryneutralizing antibodies to many different serotypes, the serotype thatwas best suited to serve as an antigen carrier in vaccine applicationsor as a therapeutic/heterologous nucleic acid carrier for gene therapyapplications was investigated. Only a few adenovirus serotypesencountered neutralizing antibodies in relatively few sera. The seraused in these studies were obtained from a large number of individualsfrom across the world, as described herein (see also, PCT InternationalPublications WO 00/70071 and WO 02/40665 and in U.S. Pat. No.6,492,169). Two adenoviruses of particular interest that encounteredantibodies in only a few sera were Ad11 and Ad35, which are bothserotypes from the B-subgroup. Generally, B-group adenovirus serotypeshave a low tropism for liver cells and are capable of efficientlyinfecting dendritic cells in vitro. In vivo studies are hampered by thefact that mice do not seem to be a proper host for subgroup Badenoviruses. Nevertheless, Ad35 has been studied in great detail andseveral recombinant derivatives based on this particular adenovirus weregenerated (PCT International Publication WO 00/70071 and in U.S. Pat.No. 6,492,169). Since Ad5-complementing cell lines were not able tosupport the growth of high titers of recombinant Ad35- andAd11-complementing cell lines, constructs and methods were alsogenerated to provide all necessary elements to generate batches ofrecombinant adenoviruses based on B-subgroup adenoviruses such as Ad35and Ad11 (U.S. Pat. No. 6,492,169). The sequences of the Ad11 and Ad35genomes were obtained in full (WO 00/70071; WO 02/53759).

Clearly, if one wants to apply a certain adenovirus serotype in avaccine composition, one should be certain that no or a low titer ofneutralizing antibodies are present in the subject that is beingtreated. It is known in the art that different levels of anti-adenovirusantibodies circulate in human individuals (D'Ambrosio et al. 1982) thatdetermine the level of therapeutic preparation that should be applied.To be able to determine in vitro the anti-adenovirus antibody titers inhuman sera, a validated adenovirus neutralization assay is required.Such a neutralization assay is also extremely useful to monitorvaccination efficiency in experimental and clinical settings and allowsstandardization. Thus, one determines the titer of neutralizingantibodies against the adenovirus serotype of interest. For this, theinvention also provides a method for determining such titers, allowingthe proper adjustment of vaccine regimens suggested by the invention. Insituations that such determinations are not feasible or easilyaccessible, for instance in mass-vaccination programs in developingcountries with poor medical infrastructure or in emergency situations,the chance of success is highest by using the serotypes disclosed hereinsince those serotypes are unlikely to encounter neutralizing activity inmost humans.

Non-limiting examples are measles, rabies virus, Ebola virus, malaria,human Metapneumovirus, etc. Antigens that could be applied are, forinstance, nucleic acids encoding measles F and H, SIV-gag,Circumsporozoite (CS) protein or LSA-1 from Plasmodium Yoelii andPlasmodium falciparum, HIV-gag/pol/nef/env, and HA and NA from Influenzavirus.

It is to be understood that differences in the capsid of the adenoviralvector would enable one to use the same backbone virus for subsequentvaccinations and prime/boost set ups, provided that the capsid ismodified by proteins that would normally be recognized by neutralizingantibodies. For instance, an Ad5 backbone carrying a fiber and/or hexonand/or a penton protein from Ad11 could be followed by a viral vectorbased on Ad5 (thus, another Ad5 backbone), wherein the capsid comprisesa fiber and/or hexon and/or a penton protein from Ad35 and vice versa.Such recombinant vectors are also encompassed by the invention. As longas the priming composition does not elicit an immune response thatsignificantly hampers the infectivity of the boosting composition (asfar as the adenoviral capsid proteins are concerned), then such primeand boost compositions are part of the invention.

The invention is further described with the aid of the followingillustrative Examples.

EXAMPLES Example 1 Low Prevalence of Neutralizing Activity to Ad11 andAd35

The analysis of neutralizing activities to Adenovirus (Ad) serotypes inhuman sera from different geographic locations (Belgium, United Kingdom,The Netherlands and two locations in the United States of America) hasbeen described elsewhere (U.S. Pat. No. 6,492,169). One of theconclusions from these studies was that neutralizing activities againstcertain adenovirus serotypes, especially Ad35 and Ad11, weresignificantly lower than those directed against Ad5.

For further analysis using serum from a location in the Far East, 100serum samples were obtained from Japan. Neutralizing activities weredetermined by the neutralization assay described in Example 1 of U.S.Pat. No. 6,492,169. A serum was set as “non-neutralizing” when, in thewell with the highest serum concentration, the protection ofcyto-pathological effect (CPE) was 90% compared to the controls withoutserum. FIG. 1 illustrates the sero-prevalence (%) related to Ad5, Ad35and Ad11, as determined in samples from Japan, Belgium, United Kingdom,The Netherlands and two locations in the United States of America. Theaverage of neutralizing activities against the three different serotypesis depicted in FIG. 2. The conclusion from this comprehensive andsystematic screening is that while more than 40% of the human seracontain neutralizing activity against Ad5, the prevalence of serumsamples neutralizing Ad11 and Ad35 is as low as 9% and 3%, respectively.These data predict that the use of adenoviral vectors based on Ad11, aswell as Ad35, will have a clear advantage over the Ad5 vectors whenexploited as vaccination vectors or gene therapy vehicles in vivo or inany application where infection efficiency is hampered by neutralizingactivity.

Furthermore, data obtained from all six different geographic locationsmentioned above, that were analyzed for Ad11-Ad35, Ad11-Ad5, Ad35-Ad5and Ad11-Ad35-Ad5 neutralization activities, showed very low percentagevalues (FIG. 3). These results strikingly highlight the differences andclearly indicate that a serum that contains antibodies directed againsta serotype from one serogroup does not necessarily contain antibodiesagainst the other serotypes from the same subgroup. This knowledgegenerates the ability to exploit combinations of Ad11- and Ad35-basedvectors (or other combinations of low-neutralized serotypes, withinsubgroups) as vaccination or gene therapy vehicles whenre-administrations or distinct vaccines are required, especially inprime/boost settings. In more advanced settings one could envisionscreening individuals for neutralizing activity against the differentserotypes and select the serotype that will encounter a low neutralizingactivity and select the prime/boost set-up that suits the treatedindividual best.

Example 2 Generation of Recombinant Adenoviral Vaccine Vectors Based onAd5

RCA-free recombinant adenoviruses can be generated very efficientlyusing adapter plasmids, such as pAdApt, and adenovirus plasmidbackbones, such as pWE/Ad.AflII-rITRsp. Methods and tools have beendescribed extensively elsewhere (U.S. Pat. Nos. 5,994,128 and 6,670,188,and International Patent Applications WO 99/55132, WO 99/64582, WO00/70071, WO 00/03029, which references are incorporated in theirentirety herein). Generally, the adapter plasmid containing thetransgene of interest in the desired expression cassette is digestedwith suitable enzymes to free the recombinant Ad sequences from theplasmid vector backbone. Similarly, the adenoviral complementationplasmid pWE/Ad.AflII-rITRsp is digested with suitable enzymes to freethe adenovirus sequences from the vector plasmid DNA.

Cloning of the gene encoding hemagglutinin from Measles virus intopIPspAdapt1.

The plasmid containing the gene encoding for the measles virushemagglutinin (MV-H) protein, pEC12/Neo/HA (De Swart et al. 1998) wasdigested with HindIII and BamHI-restriction enzymes. The 1.6 kb fragmentcorresponding to the MV-H gene was isolated from agarose gel and ligatedto HindIII and BamHI-digested pIPspAdapt1 vector (WO 99/64582). Theresulting plasmid was named pAdapt1.MV-H and contains the MV-H geneunder the transcriptional control of full-length human immediate-early(IE) cytomegalovirus (CMV) promoter and SV40 polyA(+) signal. Aschematic representation of the plasmid pAdapt1.MV-H is shown in FIG. 4.

Generation of recombinant adenovirus Ad5ΔE3.MV-H.

pAdapt1.MV-H was digested by PacI to release the left-end portion of theAd genome. Plasmid pWE.Ad.AflII-rITRspΔE3, containing the remainingright-end part of the Ad genome has a deletion of 1878 by in the E3region (XbaI deletion). This construct was also digested with PacI. BothDNAs were transfected into PER.C6™ producer cells (ECACC deposit number96022940) using lipofectamine transfection reagent (Invitrogen) asdescribed in WO 00/70071. Homologous recombination between overlappingsequences led to generation of recombinant Ad5ΔE3.MV-H. Ad vectors incrude lysates resulting from the transfections were plaque purified.Single plaques were analyzed for the presence of the transgene andamplified for large-scale production in triple-layer flasks (3×175cm²/flask). Cells were harvested at full CPE and the virus purified by atwo-step CsCl purification procedure as routinely done by those skilledin the art of adenoviral production and generally as described in U.S.Pat. No. 6,492,169.

Cloning of SIVmac239-gag into pAdapt.

The expression plasmid pcDNA31.SIVgag (GeneART) containing the codonoptimized SIVmac239 gag gene was digested with the restriction enzymesEcoRI and XBaI. The 1.56 kb fragment corresponding to the gag gene wasisolated from the agarose gel and ligated to the EcoRI and XbaI-digestedpAdapt vector (WO 00/70071). The resulting plasmid that was namedpAdapt-SIVgag contains the SIV-gag gene under the transcriptionalcontrol of the full-length CMV promoter and the SV40 polyA(+) signal. Aschematic representation of the plasmid pAdapt5-SIVgag is shown in FIG.5.

Generation of recombinant adenovirus Ad5ΔE3.SIVgag.

pAdapt5-SIVgag was digested with the restriction enzymes PacI and SalIto release the left-end portion of the Ad genome from the plasmidbackbone. The plasmid pWE.Ad.AflII-rITRΔE3, containing the remainingright-end part of the Ad genome with an 1878 by deletion in the E3region, was digested with PacI. Both DNAs are transfected intoPER-E1B55K producer cells (U.S. Pat. No. 6,492,169; also referred to asPER.C6/55K cells) using lipofectamine transfection reagent (Invitrogen).Homologous recombination between the two overlapping sequences led togeneration of recombinant Ad5ΔE3.SIVgag (generally referred to asAd5-SIVgag). Ad vectors in crude lysates resulting from thistransfection were plaque purified. Single plaques were analyzed for thepresence of the transgene and amplified for large-scale production intriple-layer flasks (3×175 cm²/flask). The culture was harvested at fullCPE and the virus purified by a two-step CsCl purification procedure anddialyzed three times into phosphate-buffered saline (PBS) containing 5%sucrose, as routinely done for adenoviruses and generally as describedin U.S. Pat. No. 6,492,169. Adenovirus titers were measured as virusparticles by HPLC using methods known to persons skilled in the art.Infectivity was measured as plaque-forming units by using PER-E1B55Kcells. SIVgag protein expression from the recombinant virus wasdetermined by infection of A549 cells followed by analysis of culturesupernatants using a commercial Gag ELISA kit (Murex Biotech, Ltd, UK).Generation of the recombinant adenovirus named Ad5ΔE3.empty (generallyreferred to as Ad5-empty) was carried out as described above, using asadapter DNA the plasmid pAdapt lacking a transgene.

Example 3 Generation of Recombinant Adenoviral Vaccine Vectors Based onAd35

RCA-free recombinant adenoviruses based on Ad35 are generated veryefficiently using adapter plasmids, such as pAdApt35Ip1 (containing Ad35nucleotides 1-464 and 3401-4669; WO 00/70071) and pAdApt535 (see below),and adenovirus plasmid backbones, such as pWE/Ad35.pIX-rITRΔE3 (U.S.Pat. No. 6,492,169).

Cloning of the measles virus hemagglutinin into pAdapt35IP1.

The plasmid containing the gene encoding for the measles virushemagglutinin (MV-H) protein pEC12/Neo/HA was digested with HindIII andBamHI. The 1.6 kb fragment corresponding to the MV-H gene was isolatedfrom agarose gel and ligated to HindIII and BamHI-digested pAdapt35IP1vector (WO 00/70071). The resulting plasmid was named pAdapt35.MV-H andcontains the MV-H gene under the transcriptional control of thefull-length human CMV promoter and SV40 polyA(+) signal. A schematicrepresentation of pAdapt35.MV-H is shown in FIG. 6.

Generation of recombinant adenovirus Ad35ΔE3.MV-H.

pAdapt35.MV-H was digested by Pad to release the Ad sequences from theplasmid backbone. Plasmid pWE.Ad35.pIX-rITRΔE3, containing the remainingright-end part of the Ad genome with 2673 by deletion in the E3 region,was digested with NotI. Both DNAs were transfected into PER-E1B55Kproducer cells using lipofectamine transfection reagent (Invitrogen).The PER-E1B55K cell line is based on PER.C6 cells that were modified bycarrying an E1B 55K gene fragment of adenovirus serotype 35, therebyenabling growth of subgroup B adenoviruses to high titers on acomplementing cell line such as PER.C6 (see for details U.S. Pat. No.6,492,169). Homologous recombination between the two overlappingsequences led to generation of recombinant Ad35ΔE3.MV-H. Ad vectors incrude lysates resulting from the transfections were plaque purified.Single plaques were analyzed for the presence of the transgene andamplified for large-scale production in triple-layer flasks (3×175cm²/flask). Cells were harvested at full CPE and the virus purified by atwo-step CsCl purification procedure as routinely done by those skilledin the art for adenoviruses and generally described in U.S. Pat. No.6,492,169.

Cloning of SIVmac239-gag into pAdapt535.

Plasmid pcDNA31.SIVgag (GeneART) containing the codon optimizedSIVmac239 gag gene was digested with the restriction enzymes EcoRI andXbaI. The 1.56 kb fragment corresponding to the gag gene was isolatedover agarose gel and ligated to the EcoRI and XbaI-digested pAdapt535vector. The resulting plasmid was named pAdapt535-SIVgag and containsthe SIV-gag gene under the transcriptional control of the full-lengthhuman CMV promoter and the SV40 polyA(+) signal. A schematicrepresentation of the plasmid pAdapt535-SIVgag is shown in FIG. 7.

Generation of recombinant adenovirus Ad35ΔE3.SIVgag.

DNA of pAdapt35-SIVgag was digested with the restriction enzymes PacI torelease the Ad sequences from the plasmid backbone. PlasmidpWE.Ad35.pIX-rITRΔE3, containing the right-end part of the Ad genomewith 2673 by deletion in the E3 region, was digested with NotI. BothDNAs were transfected into PER-E1B55K producer cells using lipofectaminetransfection reagent. Homologous recombination between the twooverlapping sequences led to generation of recombinant Ad35ΔE3.SIVgagvirus (generally referred to as Ad35-SIVgag). Adenovirus vectors incrude lysates resulting from this transfection were plaque purified.Single plaques were analyzed for the presence of the transgene andamplified for large-scale production in triple-layer flasks (3×175cm²/flask). The culture was harvested at full CPE and the virus waspurified by a two-step CsCl purification procedure and dialyzed threetimes into phosphate-buffered saline (PBS) containing 5% sucrose, asroutinely done for adenoviruses and generally as described in U.S. Pat.No. 6,492,169. Adenovirus titers were measured as virus particles byHPLC using methods known to persons skilled in the art. Infectivity wasmeasured as plaque-forming units by using PER-E1B55K cells. SIV-gagprotein expression from the recombinant virus was determined byinfection of A549 cells followed by analysis of culture supernatantsusing a commercial Gag ELISA kit (Murex Biotech, Ltd.). Generation ofthe recombinant adenovirus named Ad35ΔE3.empty was carried out asdescribed above, using as adapter DNA the plasmid pAdapt35 with notransgene.

Example 4 Recombinant Ad35ΔE3.MV-H Elicits Measles-Specific Immunity inMice Pre-Exposed to Ad5

The capacity of recombinant Ad35ΔE3.MV-H vector to induce measlesimmunity in vivo in the presence of Ad5 antibodies was investigated. Thestudy enrolled 25 Balb/C mice (female, 12 weeks old) distributed in fiveexperimental groups of five mice each. At week 0 (day 0) and week 2 (day14), mice were intravenously (i.v.) injected with either 10¹⁰ vp ofAd5Δ3-empty (groups 1 and 2) or sterile PBS (groups 3, 4 and 5) in avolume of 200 μl. Inducing pre-existing immunity may be performedthrough i.v. injections as described here, but also with intra-muscular(i.m.) injections, while all adenovirus priming and boosting injectionsfor raising an immune response are all performed with i.m. injections.At week 4 (day 28), mice belonging to groups 1 and 3 were vaccinated byintra-muscular injection of 10¹⁰ vp of Ad5ΔE3.MV-H. Similarly, mice ofgroups 2 and 4 received 10¹⁰ vp of Ad35ΔE3.MV-H in a volume of 200 μl.Also at day 28, mice of group 5 were injected with PBS (FIG. 8). Twoweeks after vaccination at day 42, cellular and humoral immunity wasdetermined by the ELISPOT and ELISA assays, respectively. Procedures forthese assays are described below. Efficient Ad35ΔE3.MV-H-mediated antimeasles-H IgG response was obtained in naïve mice (group 4) as well asin mice pre-exposed to Ad5 (group 2). In contrast, Ad5ΔE3.MV-H-inducedanti measles-H IgG response was observed only in naïve mice (group 3),whereas no significant levels of anti-measles antibodies were detectedin mice pre-exposed to Ad5 (FIG. 9). Similarly, efficient measles-HT-cell response was observed in all animals vaccinated with Ad35ΔE3.MV-Hand in the naïve mice vaccinated with Ad5ΔE3.MV-H. In contrast,measles-H T-cell response was dramatically hampered in mice exposed toAd5 prior to vaccination (FIG. 10). These results demonstrate thatAd35-based vectors can efficiently induce anti-measles immunity.Furthermore, Ad35ΔE3.MV-H-mediated anti-measles IgG and T-cell responsewas not impaired by the presence of anti-Ad5 immunity, thusstrengthening the rationale to exploit a vector based on an adenovirusagainst which the prevalence of neutralizing activity is (worldwide)low.

The ELISPOT assay was performed as follows. The mouse fibroblast cellline 3T3, syngeneic to BALB/c mice, was used as target cell line tostimulate mouse effector T-cells. At day −3 before start of the ELISPOTassay, 3T3 cells were seeded at the density of 10⁵ cells/ml in DMEMmedium, in 6-well plates, and allowed to attach to the well bottomduring 5 hours of incubation at 37° C., 10% CO₂ atmosphere. Then, Advectors were added at moi of 10⁵ vp/cell. Used vectors were Ad5.empty,Ad35.empty, Ad5ΔE3.MV-H and Ad35ΔE3.MV-H. Uninfected 3T3 were reservedfor negative controls. At day −1, multiscreen 96-well filtration plates(MAHA S45 10, Millipore) were pre-incubated with 0.5 μg/100 μl IFNγantibody (Becton Dickinson) per well, at 4° C. overnight. The next day,wells were emptied, and blocked for 1 hour at 37° C. with culture mediumIscoves supplemented with 10% FBS. At day 0, mouse splenocytes wereharvested from isolated spleen, and washed in Iscoves culture medium.Viable cells were counted by trypan blue exclusion, and cell suspensionadjusted to 10⁶ cells/ml. These “effector” spleen cells were seeded atthe density of 10⁵/100 μl/well, in the pre-coated ELISPOT plate. Targetcells were harvested and resuspended in Iscoves culture medium andadjusted to 10⁶ cells/ml. 20 U/ml of rh. IL2 (Chiron) was added totarget cell suspensions. Subsequently, 100 μl of target cells were addedin each well containing the effector cells. ELISPOT plates wereincubated overnight at 37° C., 10% CO₂. On day 1, plates were emptiedand washed six times with PBS/0.05% TWEEN, and five times with water.Second antibody, biotin rat-anti-mouse IFNγ (Becton Dickinson), wasadded to each well in 100 μl of a 2.5 μg/ml PBS/0.05% TWEEN solution.Plates were incubated for 1 hour at 37° C., and washed six times withPBS/TWEEN. Extravidine-alkaline (Millipore) was diluted 1:2000 inPBS/0.05% TWEEN/1% BSA, and added 100 μl per well. Plates were incubatedfor 1 hour at RT. A tablet of BCIP-NBT (Millipore) was dissolved in 10ml milliQ water, protected from light. Plates were washed three timeswith PBS/TWEEN and three times with PBS. The substrate BCIP-NBT solutionwas added to each well (100 μl/well), and incubated at RT. Afterapproximately 10 minutes when spots became visible, reaction was stoppedby the addition of tap water. Plates were rinsed in tap water, dried andanalyzed in an AELVIS ELISPOT reader (CLB).

The ELISA assay was generally performed as follows. High affinity ELISAplates (Greiner) were coated with inactivated measles (provided by RIVM,The Netherlands) 1:25 diluted in H₂O (50 μl per well). Plates wereincubated under UV for 1 hour. Plates were washed and blocked with 200μl PBS/1% BSA, for 1 hour at 37° C., 10% CO₂ atmosphere. Plates werewashed four times with 200 μl PBS/0.05% TWEEN. In wells 2-12, 50 μl PBSwas dispensed. In well 1, 25 μl serum and 75 μl PBS were added andserial dilutions were made by transferring 50 μl from wells 1 to 2, 2 to3, etc., through wells 11; wells 12 were left without serum. Plates andserum were incubated for 1 hour at 37° C., 10% CO₂ atmosphere. Plateswere washed four times with PBS/0.05% TWEEN 200 μl/well. To each well,50 μl of IgG-HRP (Rockland), 1:1000 diluted in PBS, were added andincubated for 1 hour at 37° C., 10% CO₂ atmosphere. Plates were washedfour times with 200 μl/well PBS/0.05% TWEEN. 100 μl/well ABTS substrate(Roche) was added, and incubated for 1 hour at 37° C., 10% CO₂atmosphere. Samples were measured for Optical Density at a wavelength of405 nm.

Example 5 Ad35ΔE3.MV-H Versus Ad5ΔE3.MV-H-Mediated Anti-Measles Responsein Cynomolgus Monkeys

The potency of the Ad35 vaccine vector in a non-human primate model wasinvestigated in an in vivo study using cynomolgus monkeys. At day 0, twomonkeys were vaccinated by intra-muscular injection with 8×10¹⁰ vp ofeither Ad35ΔE3.MV-H or Ad5ΔE3.MV-H. At day 7 and day 14post-vaccination, animal sera were collected to be analyzed for thepresence of anti-measles IgG using the MV-H-specific immunofluorescencetest as described herein. As shown in FIG. 11, both monkeys developed arelevant anti-measles antibody titer as measured on day 14post-injection with mean cellular fluorescence values of 352 (Ad5vaccinated monkey) and 626 (Ad35 vaccinated monkey), thereforedemonstrating the potency of the Ad35-based vector in a side-by-sidecomparison with the Ad5-based vector.

Example 6 Ad35ΔE3.MV-H Versus Ad5ΔE3.MV-H Anti-Measles Boost Activity inCynomolgus Monkeys Primed with Ad5ΔE3.MV-H Vaccine

The capacity of Ad35ΔE3.MV-H vector to boost measles immunity incynomolgus monkeys primed with Ad5ΔE3.MV-H vector was investigated. Thevaccination regimen was as follows: at week 0 and week 5, two monkeyswere primed with Ad5ΔE3.MV-H by intra-muscular injection of 10⁸ vp (atweek 0) and 10¹⁰ vp (at week 5). At week 25, the animals received abooster vaccination with an intra-muscular injection of 8×10¹⁰ vp ofeither Ad5ΔE3.MV-1-1 or Ad35ΔE3.MV-H. Time course of the experimentshowing time points of blood sampling and vaccination schedule isdepicted in FIG. 12. Animal sera were analyzed for the presence ofanti-MV-H antibodies using the MV-H-specific immunofluorescence test asdescribed herein. As shown in FIG. 13, relevant anti-measles immunitywas induced in the two vaccinated monkeys. However, the combination Ad5prime/Ad35 boost gave rise to the highest immuno response thusindicating the advantage offered by a prime/boost mixed-modality.

The MV-H-specific immunofluorescence test was performed as describedbelow. Pre-established Mel/Juso cell lines stably expressing the MV-Hprotein were used. Incubation of these cells with serum allows bindingof antibodies to MV-H (parental Mel/Juso cells serve as control). Afterexcess serum is removed, antibodies bound to MV-H are detected usingfluorescent-labeled antibodies specific for IgG and flow cytometry. Theassay was generally performed as follows: Mel/Juso cells stablyexpressing MV-H as well as the parental cells were seeded in 96-wellV-bottom plates at the concentration of 10⁵ cells/well in 90 μl PBSsupplemented with 3% bovine serum (P3F). Plasma samples wereheat-inactivated at 56° C. and diluted 1:10 in P3F. 10 μl of pre-dilutedplasma was added to the cells and incubated at 4° C. for 1 hour. Then,cells were washed and subsequently incubated with anti-human IgGconjugated with FITC (DAKO) diluted in 100 μl P3F. After 1 hour at 4°C., cells were washed, resuspended in 200 μl PBS and finally measured onthe FACScan.

Example 7 Comparison of Recombinant Ad5 Versus Ad35 as Vaccine Vectorsand Efficacy of Prime/Boost Regimens with Different Serotypes Ad Vectorsin Monkeys

Ad5 vectors have been demonstrated to induce effectiveanti-immunodeficiency-virus immunity (Shiver et al. 2002). Aside-by-side comparison between Ad5 and Ad35 vectors is designed toinvestigate the ability to induce immunity and protection againstimmunodeficiency virus in rhesus monkeys. In addition, the efficacy ofprime/boost regimens with different Ad vectors serotypes is evaluated.The study enrolls 24 rhesus monkeys distributed in four experimentalgroups of six monkeys each. Animals are immunized by intra-muscularinjection of 10¹¹-10¹² vp of recombinant Ad5ΔE3 or Ad35ΔE3 viral vectorscarrying either the SIVmac239 gag gene (Ad5-SIVgag or Ad35-SIVgag) or notransgene (Ad5-empty or Ad35-empty). Immunization is carried out asfollows: monkeys belonging to group 1 are vaccinated with Ad5-SIVgag atmonth 0 (prime) and month 6 (boost). Similarly, monkeys of group 2 arevaccinated with Ad35-SIVgag at month 0 (prime) and month 6 (boost).Monkeys of group 3 are vaccinated with Ad5-SIVgag at month 0 (prime) andwith Ad35-SIVgag at month 6 (boost). Finally, monkeys of group 4(control group) are vaccinated with Ad5-empty at month 0 (prime) andAd35-empty at month 6 (boost). Cellular and humoral responses aremonitored with immunological assays well known to persons skilled in theart. To evaluate efficacy of the immunity, three months after the lastvaccination, animals are challenged with 50 MD50 SHIV-89.6p virus (BethIsrael Deaconess Medical Center).

While attenuation of SHIV-89.6p virus infection in Ad5-SIVgag vaccinerecipients is predicted (Shiver et al. 2002), vaccine regimens based onAd35 alone or Ad5/Ad35 combinations are expected to be superior or atleast comparable in efficacy as compared to regimens based solely onAd5. In contrast to the findings in mice (Example 9) showing that Ad35is less potent than Ad5 in naïve mice, it is expected that Ad35 wouldelicit at least a comparable immune response in monkeys as would befound with Ad5, because the receptor-binding and/or receptor-recognitionis expected to be better in monkeys than in mice (see the discussionabout the CAR and CD46 receptors in Example 9).

Example 8 Effect of Pre-Existing Ad5 Immunity on Immunogenicity of Ad35Vaccine Vector in Monkeys

The ability of Ad35 to induce immunity and protection againstimmunodeficiency virus in rhesus monkeys in the presence of Ad5pre-existing antibodies is tested. Furthermore, the efficacy ofprime/boost regimens with different Ad vectors serotypes is evaluated.

This study enrolls 18 monkeys distributed in three experimental groupsof six monkeys each. All animals are pre-immunized at months 0 and 2with 10¹¹-10¹² vp of Ad5-empty by intra-muscular injection. At month 4(prime), monkeys of groups 1 and 3 are injected with 10¹¹-10¹² vp ofAd5-SIVgag, whereas monkeys of group 2 receive 10¹¹-10¹² vp ofAd35-SIVgag. At month 10, monkeys are boosted with 10¹¹-10¹² vp ofAd5-SIVgag (group 1) or Ad35-SIVgag (groups 2 and 3). Cellular andhumoral responses are monitored with immunological assays well known tothose skilled in the art. To evaluate efficacy of the immunity, threemonths after the last vaccination, animals are challenged with MD50SHIV-89.6p virus. It is expected that pre-existing immunity towards Ad5viruses would elicit a negative effect to a subsequent recombinant Ad5administration and thus would give less protection against a SHIVchallenge, while a subsequent recombinant Ad35 administration would notbe hindered by the pre-existing immunity that was raised to the Ad5viruses and therefore give rise to a proper protection against a SHIVchallenge.

Example 9 Effect of Pre-Existing Immunity on Immunogenicity of theAd35-SIVgag Vaccine Vector in Mice

Six to eight week-old C57/BL6 or Balb/c mice were purchased from CharlesRiver Laboratories (Wilmington, Mass.). For recombinant Ad5 and Ad35virus immunizations, mice were injected intramuscularly (i.m.) with 10⁸or 10¹⁰ vp replication-incompetent E1-deleted Ad5 or Ad35 expressingSIVmac239 Gag (SIVgag) in 100 μl sterile PBS in the quadriceps muscles.For DNA immunizations, mice were injected i.m. with 50 μg plasmidVRC-4307 expressing SIVmac239 Gag-Pol-Nef (Vaccine Research Center,National Institutes of Health) in 100 μl sterile PBS. For rMVAimmunizations, mice were injected i.p. with 10⁸ pfu rMVA-T338 expressingSIVmac239 Gag in 100 μl sterile PBS (Therion Biologics). Recombinant MVAis generally administered i.p., although other routes may be used aswell. To induce active anti-Ad5 immunity, mice were pre-immunized eitheronce or twice separated by a four-week interval i.m. with 10¹⁰ vpAd5-Empty containing no insert in 100 μl sterile PBS.

Gag-specific cellular immune responses were assessed by interferon-γ(IFN-γ) ELISPOT assays using murine splenocytes in response toindividual Gag epitope peptides or a pool of overlapping 15 amino acidpeptides covering the entire SIVmac239 Gag protein. 96-well multi-screenplates (Millipore) coated overnight with 100 μl/well of 10 μg/ml ratanti-mouse IFN-γ (Pharmingen) in PBS were washed three times withendotoxin-free Dulbecco's PBS (Life Technologies) containing 0.25%TWEEN-20 and blocked with PBS containing 5% FBS for 2 hours at 37° C.The plates were washed three times with Dulbecco's PBS containing 0.25%TWEEN-20, rinsed with RPMI 1640 containing 10% FBS, and incubated intriplicate with 2×10⁵ or 5×10⁵ splenocytes per well in a 100 μl reactionvolume containing 1 μg/ml peptide. For studies utilizing the Gag peptidepool, each peptide in the pool was present at 1 μg/ml. Following an18-hour incubation, the plates were washed nine times with Dulbecco'sPBS containing 0.25% TWEEN-20 and once with distilled water. The plateswere then incubated for 2 hours with 75 μl/well of 5 μg/ml biotinylatedrat anti-mouse IFN-γ (Pharmingen), washed six times with Coulter Wash(Coulter Corporation), and incubated for 2 hours with a 1:500 dilutionof streptavidin-AP (Southern Biotechnology Associates). Following fivewashes with Coulter Wash and once with PBS, the plates were developedwith NBT/BCIP chromogen (Pierce), stopped by washing with tap water, airdried, and read using an ELISPOT reader (Hitech Instruments). Fordepletion studies, splenocytes were incubated with magnetic microbeadscoated with anti-CD4 (L3T4) or anti-CD8 (Ly-2) mAbs (Miltenyi Biotec)and separated using MiniMACS columns prior to performing the ELISPOTassay. Cell depletions were approximately 95-98% efficient.

A direct ELISA-measured serum anti-Gag antibody titers from immunizedmice. 96-well plates coated overnight with 100 μl/well of 1 μg/mlrecombinant SIV-gag protein (Intracel) in PBS were blocked for 2 hourswith PBS containing 2% BSA and 0.05% TWEEN-20. Sera were then added inserial dilutions and incubated for 1 hour. The plates were washed threetimes with PBS containing 0.05% TWEEN-20 and incubated for 1 hour with a1:2000 dilution of a peroxidase-conjugated affinity-purified rabbitanti-mouse secondary antibody (Jackson Laboratories). The plates werethen washed three times, developed with TMB (KPL), stopped with 1% HCl,and analyzed at 450 nm with a Dynatech MR5000 ELISA plate reader.

Ad5- or Ad35-specific cellular immune responses were assessed by IFN-γELISPOT assays using murine splenocytes from C57/BL6 mice in response toAd5- or Ad35-infected syngeneic BLK CL.4 stimulator cells (ATCC TIB-81;Vogels et al. 2003). BLK CL.4 cells were plated at a density of 1×10⁶cells per well in a 6-well plate and infected with E1-deleted Ad5-Emptyor Ad35-Empty at a multiplicity of infection (moi) of 2×10⁴ for threedays. ELISPOT assays using splenocytes from immunized C57/BL6 mice werethen performed as described above using 5×10⁵ splenocytes and 1×10⁵Ad-infected BLK CL.4 stimulator cells per well in place of peptideantigens. For negative controls, splenocytes were incubated withuninfected BLK CL.4 cells or media alone.

Ad5- or Ad35-specific neutralizing antibody (NAb) responses wereassessed by luciferase-based virus neutralization assays essentially asdescribed (Vogels et al. 2003). A549 cells were plated at a density of1×10⁴ cells per well in 96-well plates. Recombinant Ad5-Luciferase orAd35-Luciferase reporter constructs were then added at an moi of 500with two-fold serial dilutions of serum in 200 μl reaction volumes.Following a 24-hour incubation, luciferase activity in the cells wasmeasured using the Steady-Glo Luciferase Reagent System (Promega). 90%neutralization titers were defined as the maximum serum dilution thatneutralized 90% of luciferase activity.

Statistical analyses were performed with GraphPad Prism version 2.01(GraphPad Software, Inc., 1996). Comparisons of mean ELISPOT responsesamong groups of mice were performed by two-tailed t tests for two groupsof animals or by analyses of variance (ANOVA) for more than two groups.Bonferroni adjustments were included when appropriate to account formultiple comparisons. In all cases, p-values of less than 0.05 wereconsidered significant.

Comparison between immunogenicity of Ad5-SIVgag and Ad35-SIVgag in naïvemice.

Groups of Balb/c and C57/BL6 mice (N=4/group) were immunized once i.m.with 10¹⁰ vp or 10⁸ vp of each vector. Vaccine-elicited cellular immuneresponses were assessed by ELISPOT assays using a pool of 15 amino acidpeptides overlapping by 11 amino acids covering the entire SIV Gagprotein. Gag-specific ELISAs assessed vaccine-elicited humoral immuneresponses. As shown in FIG. 14, both Ad5-SIVgag and Ad35-SIVgag elicitedonly marginal Gag-specific cellular immune responses in Balb/c mice. Incontrast, both vectors elicited rapid and potent cellular immuneresponses by two weeks following vaccination in C57/BL6 mice. Thedifference between the mice strains is most likely explained byrestrictions of the peptide exposure capabilities of the MHC class Imolecules in both strains. This is an antigen-specific feature. ELISPOTresponses elicited by Ad35-SIVgag were consistently lower than thoseelicited by Ad5-SIVgag, particularly at the lower dose of 10⁸ vp. Hightiter anti-Gag antibody responses were elicited by Ad5-SIVgag in bothBalb/c and C57/BL6 mice. In contrast, no anti-Gag antibody responseswere detected following immunization with Ad35-SIVgag. Thus, Ad35-SIVgagelicited lower cellular and humoral immune responses as compared withAd5-SIVgag in naïve mice. A reason for this finding may be thedifference in receptor-binding and/or -recognition in mice as comparedto humans (see below).

Mapping D^(b)-restricted T-lymphocyte epitopes within SIV Gag.

The rapid emergence of high frequency Gag-specific cellular immuneresponses in C57/BL6 mice (FIG. 14, Panel C) suggested the presence ofimmunodominant D^(b)- or K^(b)-restricted CD8⁺ T-lymphocyte epitopes.Therefore a matrix-based ELISPOT approach was utilized to identifycandidate epitopes within SIV-Gag. As depicted in FIG. 15, Panels A-C,C57/BL6 mice immunized with 10¹⁰ vp recombinant Ad5, 10¹⁰ vp recombinantAd35, or 50 μg plasmid DNA expressing SIV-Gag developed animmunodominant cellular immune response to the 15 amino acid P78 peptide(QTDAAVKNWMTQTLL; SEQ ID NO:1) and a subdominant response to the P19peptide (ENLKSLYNTVCVIWC; SEQ ID NO:5). ELISPOT assays utilizingsplenocytes depleted of CD4⁺ or CD8⁺ T-lymphocytes demonstrated thatboth P78 and P19 were in fact CD8⁺ T-lymphocyte epitopes.

Next, these epitopes based on the peptide-binding motifs of D^(b)(asparagine at position 5 and hydrophobic carboxy-terminus) and K^(b)(tyrosine at position 5 and hydrophobic carboxy-terminus) werefine-mapped. As shown in FIG. 15, Panels D-F, candidate optimal peptideswere assessed at log dilutions from 1 μg/ml to 100 fg/ml inpeptide-specific ELISPOT assays. A D^(b)-restricted immunodominant AL11(AAVKNWMTQTL; SEQ ID NO:4) epitope within P78 and a D^(b)-restrictedsubdominant KV9 (KSLYNTVCV; SEQ ID NO:6) epitope within P19 wereidentified. These CD8⁺ T-lymphocyte epitopes elicited ELISPOT responseswhen utilized at concentrations of 1 pg/ml and were confirmed byfunctional chromium-release cytotoxicity assays using peptide-pulsed EL4cells as well as Ltk cells transfected with D^(b), but not Ltk cellstransfected with K^(b), as targets. The LV10 peptide was similarlyinvestigated as a potential K^(b)-restricted epitope, but this peptidecould not be confirmed in cytotoxicity assays using Ltk cellstransfected with K^(b) as targets, suggesting that its reactivity inELISPOT assays may reflect KV9 contaminant peptide within the LV10preparation.

Ad5-specific and Ad35-specific NAb titers in humans.

Next, 12 human serum samples for NAb titers to Ad5 and Ad35 wereassessed. 90% NAb titers were defined as the maximal serum dilution thatinhibited rAd5-Luciferase or rAd35-Luciferase infectivity of A549 cellsby 90%. As shown in FIG. 16, eight of twelve samples (67%) exhibitedAd5-specific NAb titers of 32 or higher. In addition, two of twelvesamples (17%) had Ad5-specific NAb titers of 2048 or higher. None ofthese samples had detectable Ad35-specific NAb titers (<16). These dataare consistent with previous observations regarding the highseroprevalence of Ad5 and the low seroprevalence of Ad35 (Vogels et al.2003). Positive controls were sera from mice immunized with Ad5 or Ad35.

Immunogenicity of Ad5-SIVgag and Ad35-SIVgag in mice with anti-Ad5immunity.

The impact of anti-Ad5 immunity on cellular immune responses elicited byAd5-SIVgag and Ad35-SIVgag was determined. To model anti-Ad5 immunity,C57/BL6 mice were pre-immunized once with 10¹⁰ vp rAd5-Empty at fourweeks prior to immunization. As shown in FIG. 17, Panel C, micepre-immunized with Ad5-Empty developed mean Ad5-specific neutralizingantibody (NAb) titers of 128 but no detectable Ad35-specific NAb titers(<16). These NAb titers were comparable with those typically found inhumans (FIG. 16). Ad-specific T-lymphocyte responses in these mice wereassessed by virus-specific ELISPOT assays using splenocytes stimulatedwith Ad5- or Ad35-infected syngeneic BLK/CL.4 cells. As shown in FIG.17, Panel D, mice pre-immunized with Ad5-Empty developed Ad5-specificELISPOT responses of 250 SFC/10⁶ splenocytes but no detectableAd35-specific ELISPOT responses (<25 SFC/10⁶ splenocytes).

Groups of naïve mice or mice with anti-Ad5 immunity (N=4/group) werethen immunized with 10¹⁰ vp or 10⁸ vp Ad5-SIVgag or Ad35-SIVgag. Fourweeks following immunization, vaccine-elicited cellular immune responseswere assessed by Gag pooled peptide and epitope-specific ELISPOT assays.As shown in FIG. 17, Panels A and B, Gag-specific and epitope-specificcellular immune responses elicited by 10¹⁰ vp Ad5-SIVgag were blunted by75% in mice with anti-Ad5 immunity as compared with naïve mice. Cellularimmune responses elicited by 10⁸ vp Ad5-SIVgag were completely abrogatedin mice with anti-Ad5 immunity. In contrast, responses elicited byAd35-SIVgag were not substantially reduced in mice with anti-Ad5immunity and were higher than those elicited by Ad5-SIVgag (p<0.05comparing pooled peptide ELISPOT responses using two-tailed t tests).

Vector-specific humoral and cellular immune responses were also assessedin these groups of mice. As shown in FIG. 17, Panel C, naïve miceimmunized with Ad5-SIVgag or Ad35-SIVgag developed Ad serotype-specificNAb responses. As expected, mice pre-immunized with Ad5-Empty generatedpotent, anamnestic (secondary) Ad5-specific NAb responses followingAd5-SIVgag immunization. Interestingly, mice pre-immunized withAd5-Empty also generated potent, anamnestic Ad35-specific NAb responsesfollowing Ad35-SIVgag immunization. These responses were >10-fold higherthan the Ad35-specific NAb responses generated in naïve mice followingthe same Ad35-SIVgag immunization, suggesting that pre-immunization withAd5-Empty may have primed for low levels of cross-reactive Ad5/Ad35responses. As shown in FIG. 17, Panel D, naïve mice immunized withAd5-SIVgag or Ad35-SIVgag developed Ad serotype-specific ELISPOTresponses, and mice pre-immunized with Ad5-Empty generated higherAd5-specific ELISPOT responses following Ad5-SIVgag immunization.

Immunogenicity of Ad5-SIVgag and Ad35-SIVgag in mice with high levels ofanti-Ad5 immunity.

Next, the ability of high levels of anti-Ad5 immunity to suppresscellular immune responses elicited by Ad5-SIVgag and Ad35-SIVgag wasassessed. Mice were pre-immunized twice with 10¹⁰ vp Ad5-Empty at eightweeks and four weeks prior to immunization. As shown in FIG. 18, PanelC, these mice developed mean Ad5-specific NAb titers of 16,384 but nodetectable Ad35-specific NAb titers (<16). As shown in FIG. 18, Panel D,these mice also developed high frequency Ad5-specific ELISPOT responsesof 560 SFC/10⁶ splenocytes but no detectable Ad35-specific ELISPOTresponses (<25 SFC/10⁶ splenocytes). Thus, pre-immunization of mice withtwo doses of Ad5-Empty generated >10-fold higher Ad5-specific NAb titersand >2-fold higher Ad5-specific ELISPOT responses as compared withpre-immunization of mice with one dose of Ad5-Empty.

Groups of naïve mice or mice with high levels of anti-Ad5 immunity(N=4/group) were then immunized with 10¹⁰ vp Ad5-SIVgag or Ad35-SIVgag.Four weeks following immunization, Gag-specific cellular immuneresponses were assessed. As shown in FIG. 18, Panels A and B, highlevels of anti-Ad5 immunity abrogated Gag-specific and epitope-specificELISPOT responses elicited by 10¹⁰ vp Ad5-SIVgag by >90%, whereasELISPOT responses elicited by 10¹⁰ vp Ad35-SIVgag were at mostmarginally reduced. Thus, in mice with high levels of anti-Ad5 immunity,Ad35-SIVgag was substantially more immunogenic than Ad5-SIVgag (p<0.001comparing pooled peptide and peptide-specific ELISPOT responses usingtwo-tailed t tests). As shown in FIG. 18, Panels C and D, Ad5-specificNAb responses and Ad5-specific ELISPOT responses in mice with highlevels of anti-Ad5 immunity were not further increased followingAd5-SIVgag immunization. These data suggest that the Ad5-SIVgag vaccinevector was rapidly neutralized before eliciting substantialantigen-specific or vector-specific immune responses in these mice.

Immunogenicity of heterologous prime/boost regimens in naïve mice.

Next, the immunogenicity of several heterologous prime/boost regimens innaïve C57/BL6 mice was assessed. Groups of mice (N=4/group) were primedwith either 50 μg plasmid DNA, 10¹⁰ vp recombinant Ad5, 10¹⁰ vprecombinant Ad35, or 10⁸ pfu recombinant MVA all expressing SIV-Gag.Mice were then boosted at week 4 with either 10¹⁰ vp recombinant Ad5,10¹⁰ vp recombinant Ad35, or 10⁸ pfu recombinant MVA and sacrificed atweek 8 for immunologic assays. As shown in FIG. 19, Panel A, plasmid DNAexpressing SIV-Gag primed low frequency Gag-specific cellular immuneresponses. These responses were boosted effectively and comparably byAd5-SIVgag, Ad35-SIVgag, and rMVA-Gag. In contrast, Ad5-SIVgag primedhigher levels of Gag-specific cellular immune responses, but theseresponses were not significantly boosted by a second administration ofAd5-SIVgag, presumably as a result of anti-Ad5 immunity. Importantly,responses primed by Ad5-SIVgag were boosted effectively by Ad35-SIVgag.These data demonstrate that Ad35-SIVgag is efficient at boostingresponses primed by DNA-Gag or Ad5-SIVgag. These data further suggestthat prime/boost regimens utilizing heterologous adenovirus vectors canelicit potent immune responses comparable in magnitude to those elicitedby DNA prime-viral vector boost regimens.

The Ad35-SIVgag and rMVA-Gag vectors primed moderate levels ofGag-specific cellular immune responses. Interestingly, the recombinantAd5 prime-recombinant Ad35 boost regimen was substantially moreimmunogenic than the recombinant Ad35 prime-recombinant Ad5 boostregimen. Moreover, the recombinant Ad5 prime-recombinant MVA boostregimen was slightly more immunogenic than the recombinant MVAprime-recombinant Ad5 boost regimen. These results suggest that theorder of vector administration may be important to achieve optimalimmunogenicity. Thus, the more potent priming vector is preferably butnot necessarily used to prime the response and the more potent boostingvector is preferably but not necessarily used to boost the response whentwo recombinant viral vectors are utilized in prime/boost regimens. Itis to be understood that this effect may be antigen specific. For oneapplication applying a certain antigen, one serotype may be the morepotent prime or boost vector, while this may be different in anotherapplication applying another antigen. The invention therefore alsorelates to methods for addressing what vector is the more potent vectorduring priming or boosting, using different antigens.

As shown in FIG. 19, Panel B, mice primed with Ad5-SIVgag and boostedwith Ad35-SIVgag developed >10-fold higher Ad35-specific NAbs ascompared with naïve mice primed with Ad35-SIVgag. Similarly, mice primedwith Ad35-SIVgag and boosted with Ad5-SIVgag developed >10-fold higherAd5-specific NAbs as compared with naïve mice primed with Ad5-SIVgag.These data suggest that either low levels of cross-reactive NAbs(titers<16) or helper T-lymphocyte responses were primed by each Advector and resulted in anamnestic responses following administration ofthe heterologous Ad vector. In a subsequent administration it wouldtherefore be preferred to use another vector (which is heterologous tothe previous) to obtain a sufficiently high immune response. This wouldfor instance be the case for vaccination situations in which severalboosts are required or in cases wherein several different antigens areto be delivered over time.

Immunogenicity of heterologous prime/boost regimens in mice withanti-Ad5 immunity.

Since the majority of humans have pre-existing anti-Ad5 immunity, it isimportant to assess candidate heterologous prime/boost regimens inanimals with anti-Ad5 immunity. Therefore, the immunogenicity of variousprime/boost regimens in C57/BL6 mice that were pre-immunized once with10¹⁰ vp rAd5-Empty at 4 weeks prior to primary immunization weredetermined. These mice had Ad5-specific NAb titers of 128-256 at thetime of primary immunization (FIG. 20, Panel C). Groups of mice(N=4/group) were primed at week 0 with 50 μg DNA or 10¹⁰ vp recombinantAd5 expressing SIV-Gag and then boosted at week 4 with 10¹⁰ vprecombinant Ad5, 10¹⁰ vp recombinant Ad35, or 10⁸ pfu recombinant MVAexpressing SIV-Gag. Mice were sacrificed at week 8 for immunologicassays.

As shown in FIG. 20, Panels A and B, the DNA prime-recombinant Ad35boost regimen elicited higher ELISPOT responses than the recombinant Ad5prime-recombinant Ad35 boost or the recombinant Ad5 prime-recombinantMVA boost regimens (p<0.01) and markedly higher ELISPOT responses thanthe DNA prime-recombinant Ad5 boost regimen in these mice (p<0.001comparing pooled peptide and dominant epitope-specific ELISPOT responsesamong groups of mice using ANOVA with Bonferroni adjustments to accountfor multiple comparisons). Thus, in mice with anti-Ad5 immunity, the DNAprime-recombinant Ad35 boost regimen elicited the highest frequencyELISPOT responses among these various regimens with Gag pooled peptideresponses of >1500 SFC/10⁶ splenocytes and P78- and AL11-specificresponses of >2500 SFC/10⁶ splenocytes.

Thus, it was shown that anti-Ad5 immunity markedly blunted theimmunogenicity of recombinant Ad5-SIVgag. In contrast, even high levelsof anti-Ad5 immunity did not substantially reduce the immunogenicity ofrecombinant Ad35-SIVgag in mice. In particular, in mice with anti-Ad5NAb titers comparable with those typically found in humans, cellularimmune responses elicited by Ad35-SIVgag were higher than those elicitedby Ad5-SIVgag.

In naïve mice, however, Ad35-SIVgag elicited substantially lowercellular and humoral immune responses than did Ad5-SIVgag. Thesedifferences in immunogenicity are consistent with previous observationsthat Ad35-mediated transgene expression was several-fold lower thanAd5-mediated transgene expression in mouse muscle (Vogels et al. 2003).In addition, Ad5 interacts with the Coxsackievirus and AdenovirusReceptor (CAR) on the surface of cells with its long and flexible fiberprotein. In contrast, the Ad35 fiber protein is shorter and more rigidthan the Ad5 fiber, and its receptor is distinct from CAR. It wasrecently shown that B-group adenoviruses recognize the CD46 protein onthe cell surface (Gagger et al. 2003; Segerman et al. 2003). As aresult, Ad5 and Ad35 (and Ad11, being a subgroup B virus) exhibitdifferent cellular tropisms. For example, Ad35 infects human dendriticcells, smooth muscle cells, and synoviocytes more efficiently than Ad5.Moreover, Ad5 and Ad35 have different intracellular trafficking pathwaysand escape endosomes at different stages (Shayakhmetov et al. 2003).These differences in attachment and intracellular trafficking likelyaccount in part for the differences in immunogenicity observed withAd5-SIVgag and Ad35-SIVgag. At present, it is not clear why thedifferences in Gag-specific humoral immune responses between Ad5-SIVgagand Ad35-SIVgag were far more striking than the differences inGag-specific cellular immune responses between these two vectors. It ispossible that a higher threshold of antigen is needed to generateantibody responses as compared with T-lymphocyte responses in thissystem.

The heterologous prime/boost experiments demonstrated potent Ad-specificNAb responses in mice following priming and boosting with heterologousAd vectors. It is possible that cross-reactive NAb responses below thelimit of detection (titers<16) were elicited by each Ad vector and wererecalled following administration of the heterologous Ad vector. Analternate possibility is that cross-reactive helper T-lymphocyteresponses were elicited by each Ad vector and led to robust NAbresponses following administration of the heterologous Ad vector.Regardless, the immunogenicity of Ad35-SIVgag was not substantiallyblunted in mice with anti-Ad5 immunity despite the rapid evolution ofhigh titer anti-Ad35 NAb responses. These data show that anti-vector NAbresponses present at the time of immunization may be more important thanthose that develop following immunization in determining their potentialsuppressive effects on vaccine immunogenicity.

The heterologous prime/boost studies further demonstrated thatrecombinant Ad35 vectors efficiently boosted cellular immune responsesprimed by plasmid DNA and Ad5 vaccines in mice both with and withoutanti-Ad5 immunity. In mice with anti-Ad5 immunity, the DNAprime-recombinant Ad35 boost regimen was significantly more immunogenicthan the DNA prime-recombinant Ad5 boost, recombinant Ad5prime-recombinant MVA boost, and recombinant Ad5 prime-recombinant Ad35boost regimens. It appears that Ad35-SIVgag may be less effective thanAd5-SIVgag at priming immune responses in mice but at least as effectiveat boosting immune responses, although it cannot be excluded that this“better boost” effect is antigen specific.

Example 10 Construction of a Plasmid-Based System to Generate Ad11Recombinant Viruses

In order to construct the Ad11 adapter plasmid pAdApt11 the necessaryAd11 sequences were PCR-amplified from wild-type Ad11 viral DNA andcombined with the expression cassette containing a promoter, polylinkerand poly-adenylation signal taken from pAdAptp35IP1 (described in detailin WO 00/70071).

Wild-type Ad11 viruses (RIVM, The Netherlands) were propagated onPER.C6™ cells and DNA was isolated from 300 μl of purified virus(1.5×10¹² vp/ml) using phenol/chloroform extraction, ethanolprecipitation and ethanol (70%) wash procedures known to the personskilled in the art and generally as described in WO 00/70071. The entireAd11 sequence was obtained from a shotgun library generated fromrandomly sheared DNA that was blunt-ended with the Klenow enzyme (NewEngland Biolabs). Blunt-ended fragments (1-3 kb in length) were purifiedfrom a low-melting point agarose gel. The shotgun library wasconstructed after ligation of these purified size fractionated DNAfragments into the SmaI site of the pUC19 cloning vector and amplifiedon competent XL1-Blue MRF′ bacteria (Stratagene). After libraryamplification transformed bacteria were plated on LB-agar platescontaining ampicillin, X-gal and IPTG. An array of clones in 96-wellplates covering the Ad11 genome 8 times was used to generate the entiresequence. DNA sequencing was performed using Big Dye Terminatorchemistry, with AmpliTaq FS DNA polymerase using Puc forward or reversesequencing primers. Reactions were analyzed on ABI3100 and ABI3700sequencers. In total there were 687 sequencing reads obtained which wereassembled into the final contig using the Phred-Phrap software package.The resulting contig covers the entire Ad11 genome, being 34,794 basepairs in length (WO 02/53759).

The Ad11 left Inverted Terminal Repeat (left ITR or IITR) and packagingsequence (corresponding to wt-Ad11 sequence 1-464) was PCR-amplifiedfrom wt-Ad11 DNA template using primers 35F1 and 35R2 (for reference tothe sequence of these primers, see WO 00/70071). PCR-amplificationintroduces a PacI site at the 5′ end and an AvrII site at the 3′ end ofthe amplified product. For the amplification reactions Pwo DNApolymerase enzyme (Roche) was used according to manufacturer'sinstructions, DMSO was added to a final concentration of 3% and 0.6 mMof both forward and reverse primers were used. The amplification programwas set as follows: 2 minutes at 94° C., 30 cycles of: 30 seconds at 94°C., 30 seconds at 60° C. and 1 minute at 72° C.; followed by one finalextension of 8 minutes at 68° C. The amplified DNA product was purifiedusing the Qiaquick PCR purification kit (Qiagen) according to themanufacturer's instructions. The fragment was then cloned into the SrfIsite of the pre-digested pPCRScript.Amp(SK+) cloning vector (Stratagene)and grown in DH5α-competent (max. efficiency) bacteria (Invitrogen). Theresulting plasmid was named pPCRScript.11TR and was analyzed byrestriction enzyme digestions using BssHII, AccI and AvrII/PacI. Onepositive clone, with the insert in the correct orientation, wasselected, grown and digested sequentially with AvrII and SacII. Theclones with correct orientation yielded a 439 by fragment afterdigestion with AccI. The double-digested vector was recovered fromagarose gel and purified using the Qiaquick gel extraction kit (Qiagen)and used in further cloning procedures (see below).

Next, the part of the Ad11 genome downstream of the El region andcorresponding to wt-Ad11 sequence 3400 to 4670, was generated. Thisregion harbors the sequence mediating homologous recombination andgeneration of recombinant Ad11 viruses when used with a cosmid carryingthe Ad11 genome from the pIX coding region towards the right ITR (rITR).The entire 1.27 kb fragment was generated with PCR-amplification usingwt-Ad11 DNA as a template using the primers 35F3 and 35R4 (for referenceto the sequence of the applied primers, see WO 00/70071).PCR-amplification introduces a BglII site at the 5′ end and a PacI siteat the 3′ end of the sequence. PCR procedures were performed asdescribed above. The amplified DNA product was purified using theQiaquick PCR purification kit. The purified fragment was then clonedinto the SrfI site of pre-digested and gel-purified pPCRScript.Amp(SK+)cloning vector, using methods known to persons skilled in the art. Theresulting plasmid was named pPCRScript.overlap and further digested withBglII and SacII-restriction enzymes. The resulting 1.27 kb BglII-SacIIfragment that represents the overlapping Ad11 DNA was isolated overagarose gel.

Plasmid pAdApt35IP1 (WO 00/70071) was digested with AvrII andBglII-restriction enzymes. The 4.34 kb AvrII-BglII fragment,representing the CMV promoter, the multiple cloning site and the SV40poly(A) tail was isolated and purified. This AvrII-BglII fragment, theisolated BglII-SacII fragment from pPCRScript.overlap (see above) andthe isolated SacII-AvrII-digested pPCRScript.11TR (see above) werecloned together in a three-point ligation procedure using methods knownto persons skilled in the art. The resulting plasmid was namedpPCRScript-Ad11 (FIG. 21). Subsequently, pPCRScript-Ad11 was digestedwith PacI-restriction enzyme and the resulting 2.8 kb fragment wasisolated over agarose gel. This 2.8 kb fragment was then cloned into thevector fragment of pAdApt35IP1 following digestion with PacI andisolation from gel. The resulting vector was named pAdApt11 (FIG. 22).

Except for a HindIII site in the polylinker, pAdApt11 contained a secondHindIII site at position 3934 (calculated from the Ad11 genome) in theoverlap sequence that is required for homologous recombination. In orderto have a unique HindIII site in the polylinker, the second HindIII sitewas eliminated. For this, pAdApt11 was partially digested with HindIII.The protruding HindIII ends were filled-in with dNTPs using Klenowenzyme. The partially digested vector was re-circularized andtransformed into DH5α-competent bacteria. The resulting construct wasnamed pAdApt11.dH (FIG. 23).

The part of the Ad11 genome downstream of the E1 region, lacking the pIXpromoter and corresponding to wt-Ad11 sequence 3466 to 4668, was alsogenerated. This region of the genome also harbors an overlapping part ofthe Ad11 genome for proper homologous recombination and generation ofrecombinant Ad11 viruses when used with a cosmid carrying the rest ofthe Ad11 genome towards the right ITR (rITR). First, a 1.2 kb fragment(nucleotides 3465-4668) was PCR-amplified using pAdApt11.dH as atemplate with the primers pIX11Fmfe (5′-CTC TCT CAA TTG TCT GTC TTG CAGCTG TCA TG-3′ SEQ ID NO:10) and 35R4 (for reference to the sequence ofthe 35R4 primer, see WO 00/70071). The PCR-amplification introduces anMfeI site at the 5′ end of the fragment. An ApaI site is internallypresent in the amplified product.

For the PCR 2.5 U of Pfu DNA polymerase enzyme (Promega) was used, whilethe following PCR program was applied: 3 minutes at 94° C.; 5 cycles of30 seconds at 94° C., 30 seconds at 56° C. and 2 minutes at 72° C.; 25cycles of 30 seconds at 94° C., 30 seconds at 60° C., 2 minutes at 72°C.; and one single final extension of 8 minutes at 68° C. The amplifiedDNA product was purified using the Qiaquick PCR purification kit(Qiagen) and fragments were then cloned into the SrfI site of apre-digested pPCRScript.Amp(SK+) cloning vector. The resulting plasmidwas designated pPCRScript.overlap.dPr and subsequently digested withMfeI and ApaI. This MfeI-ApaI-restriction fragment was recovered from anagarose gel.

A first 101 by PCR fragment containing the Ad5 pIX promoter (nucleotides1509-1610 was generated with the primers SV40for (5′-CAA TGT ATC TTA TCATGT CTA G-3′ SEQ ID NO:11) and pIXSRmfe (5′-CTC TCT CAA TTG CAG ATA CAAAAC TAC ATA AGA CC-3′ SEQ ID NO:12). The reaction was done with Pwo DNApolymerase according to manufacturer's instructions but with 3% DMSO inthe final mix. pAdApt (see WO 00/70071) was used as a template. Theprogram was set as follows: 2 minutes at 94° C.; 30 cycles of: 30seconds at 94° C., 30 seconds at 52° C. and 30 seconds at 72° C.;followed by 8 minutes at 72° C. The resulting PCR fragment contains the3′ end of the SV40 polyadenylation signal from pAdApt and the Ad5-pIXpromoter region as present in GenBank Accession number M73260 fromnucleotide 3511 to nucleotide 3586 and an MfeI site at the 3′ end. Asecond PCR fragment was generated as described above but with primerspIX35Fmfe (5′-CTC TCT CAA TTG TCT GTC TTG CAG CTG TCA TG-3′ SEQ IDNO:13) and 35R4. pAdApt35IP1 was used as a template, the annealing wasset at 58° C. for 30 seconds and the elongation of the PCR program wasset at 72° C. for 90 seconds. This PCR procedure amplifies Ad35sequences from nucleotide 3467 to nucleotide 4669 (sequence numbering asin WO 00/70071) and adds an MfeI site to the 5′ end. Both PCR fragmentswere digested with MfeI and purified using the Qiagen PCR purificationkit (Qiagen). Approximate equimolar amounts of the two fragments wereused in a ligation reaction. Following an incubation of two hours withligase enzyme in the correct buffers, at room temperature, the mixturewas loaded on an agarose gel and the DNA fragments of 1.4 kb length wereisolated with the Geneclean II kit (BIO101, Inc). The purified DNA wasused in a PCR amplification reaction with primers SV40 and 35R4. The PCRwas done as described above with an annealing temperature of 52° C. andan elongation time of 90 seconds. The resulting product was isolatedfrom gel using the Qiagen gel extraction kit and digested with AgeI andBglII. The resulting 0.86 kb fragment containing the complete 100nucleotide pIX promoter form Ad5, the MfeI site and the pIX ORF(fragment MfeI-AgeI, including the ATG start site) from Ad35, butwithout a poly(A) sequence, was isolated from gel using the Geneclean IIkit.

pAdApt35.Luc (described in WO 00/70071) was also digested with BglII andAgeI and the 5.8 kb vector was isolated from gel using the Geneclean IIkit as described above. This fragment was ligated with the isolated 0.86kb BglII-AgeI fragment containing the Ad5-Ad35 chimeric pIX promoter, toresult in a plasmid named pAdApt535.Luc (FIG. 24).

pAdApt535.Luc was subsequently digested with BglII and ApaI and the 1.2kb insert was purified from gel. pAdApt35IP1 was also digested withBglII and ApaI and the 3.6-kb vector fragment was isolated as above.Ligation of the 1.2 kb BglII-ApaI insert from pAdApt535.Luc and the 3.6kb BglII-ApaI-digested vector resulted in pAdApt535 (FIG. 25). Thus,pAdApt535 is an Ad35 adapter plasmid containing part of the Ad5-pIXpromoter sequence but is otherwise identical to Ad35 adapter plasmidpAdApt35IP1 (see WO 00/70071). pAdApt535 was then digested with MfeI andApaI. The digested vector was recovered from an agarose gel usingGeneclean II.

The MfeI-ApaI fragment of pPCRScript.overlap.dPr (described above) wascloned into an MfeI-ApaI-digested pAdApt535 vector (see above), usinggeneral molecular biology methodology. The resulting vector was namedpAdApt535.overlap.dPr11 (FIG. 26). This vector contains the left ITR ofAd35, an expression cassette and contains the Ad11 overlap regionlacking the original Ad11 pIX promoter. The pIX promoter from Ad11 hasbeen exchanged for the Ad5-pIX promoter. The reason for constructingsuch plasmid was that it served as an in-between construct through whichpart of the Ad11 overlap (containing the Ad5-pIX promoter: theEcoNI-BglII fragment) was used to generate pAdApt511. The whole overlap(ApaI-BglII fragment) was used to generate pAdApt511.dH (see below).

pAdApt535.overlap.dPr11 was digested with ApaI and gel-purified. Linearvector DNA was then digested with BglII. The insert (1253 bp) wasgel-purified. Another digestion was performed on the same plasmid withEcoNI and BglII generating a 389 by insert which was also gel-purified.

Plasmid pAdApt11.dH was digested with ApaI-BglII, and another aliquot ofthe same plasmid was digested with EcoNI-BglII. The ApaI-BglII insert(1249 bp) from pAdApt535-overlap.dPr11 was ligated into theApaI-BglII-digested pAdApt11.dH vector resulting in pAdApt511.dH (FIG.27), while the EcoNI and BglII insert (389 bp) frompAdApt535-overlap.dPr was cloned into the EcoNI and BglII-digestedpAdApt11 vector resulting in pAdApt511 (FIG. 28).

Generation of Ad11-Based Cosmid Clones

To obtain the Ad11 sequences corresponding to positions 3400 to 6770 ofthe Ad11 genome, a PCR amplification was carried out using Pwo DNApolymerase and wt-Ad11 DNA as a template with primers 35F5 and 35R6 (forreference to the primer sequences, see WO 00/70071). ThisPCR-amplification introduces a NotI site at the 5′ end of the amplifiedproduct. The amplification program was set as follows: 2 minutes at 94°C.; 30 cycles of: 30 seconds at 94° C., 30 seconds at 65° C., 1 minute45 seconds at 72° C.; 8 minutes at 68° C. The amplified DNA product waspurified using the Qiaquick PCR purification kit (Qiagen). This 3.3 kbfragment was then cloned into an SrfI-digested pPCRScript.Amp(SK+). Theresulting plasmid was named pPCRScript.pIX and contains the pIX gene andpromoter of Ad11. Next, the plasmid was digested with NotI and NdeI.This NotI-NdeI-restriction fragment was gel-purified.

To generate a fragment containing the Ad11 genomic sequences fromnucleotide 33095 to 34794 (including the right ITR), another PCRamplification was performed using Pwo DNA polymerase and wt-Ad11 DNA asa template and with 35F7 and 35R8 primers (for reference to the sequenceof the primers, see WO 00/70071). This PCR-amplification introduces aNotI site at the 3′ end of the amplified product. The amplificationprogram was set as follows: 3 minutes at 94° C.; 5 cycles of: 30 secondsat 94° C., 45 seconds at 40° C., 2 minutes 45 seconds at 72° C.; 25cycles of: 30 seconds at 94° C., 30 seconds at 60° C. and 2 minutes 45seconds at 72° C.; 8 minutes at 68° C. The amplified DNA product (1.7kb) was purified using the Qiaquick PCR purification kit and cloned intoan SrfI-digested pPCRScript.Amp(SK+), resulting in a plasmid namedpPCRScript.rITR. This plasmid was subsequently digested with NotI andNdeI, resulting in a 1.7 kb fragment that was recovered from an agarosegel using Geneclean II.

To generate a vector containing the pIX promoter region of Ad11 as wellas the rITR of Ad11, first cosmid vector pWE15 (Clontech Laboratories,Inc.) was digested with NotI, de-phosphorylated using Calf IntestinalPhosphatase (New England Biolabs) and purified from gel using GenecleanII. Then, the NotI-NdeI fragment from pPCRScript.pIX and the NotI-NdeIfragment from pPCRScript.rITR were simultaneously ligated in athree-point ligation procedure into the NotI-digested cosmid pWE15,using methods generally applied by persons skilled in the art. Theligation mixture was transformed into STBL2-competent bacterial cells.After restriction analyses with NcoI and NdeI/HindIII-restrictionenzymes, it turned out that there were no vectors having the insert inthe correct orientation, but in contrast the insert was present in theantisense orientation (rITR-pIX). To obtain a construct containing theinsert in the desired orientation, the NotI fragment NotI-rITR-pIX-NotIwas cut from the wrong-orientation vector, purified over gel and clonedinto a fresh pWE15 vector digested with NotI. Restriction analyses onDNA mini-preparations (Qiaprep spin miniprep kit) with HindIII yieldedconstructs that harbored inserts in the correct orientation(NotI-pIX-rITR-NotI). The resulting vector was named pWE.Ad11.dNdeI(FIG. 29), wherein dNdeI refers to the deletion of the 26.6 kb NdeIfragment, not to the NdeI site.

Sequences starting just downstream of the Ad11 pIX promoter region werePCR-amplified with pfu polymerase. The region that is amplified herecorresponds to nucleotide 3480 to 4658. Two separate templates wereused: pWE.Ad11.dNdeI FIG. 29) and pAdApt11.dH (FIG. 23). Both templateswere used in amplifications using the same primer pair: Ad11 pIXcos(2)(5′-CTG CTG GAC GTC GCG GCC GCG TCA TGA GTG GAA ACG CTT C-3′ SEQ IDNO:14) and 35R3 (for reference to the sequence of 35R3, see WO00/70071). PCR introduced an AatII site at the 5′ site of the fragmentwhile an AgeI site was internally present within the sequence. Theamplification program was set as follows: 3 minutes at 94° C.; 5 cyclesof: 30 seconds at 94° C., 30 seconds at 56° C., 2 minutes at 72° C.; 25cycles of: 30 seconds at 94° C., 30 seconds at 60° C., 2 minutes at 72°C.; 8 minutes at 68° C. The amplified DNA products (1.2 kb) werepurified using the Qiaquick PCR purification kit. Both the 1.2 kbfragment obtained after PCR on pWE.Ad11.dNdeI, as well as the 1.2 kbfragment obtained after PCR on pAdApt.11.dH, were cloned separately intoSrfI-digested pPCRScript.Amp(SK+). The resulting plasmids were namedpPCRScript.pIX.dPr and pPCRScript.pIX.dH.dPr, respectively. Bothconstructs were digested with AatII and AgeI. The AatII-AgeI-restrictionfragments represent a part of the pIX-ORF start with the ATG at position3483, to position 4658. The fragments just start downstream of the pIXpromoter and have the AgeI-restriction site at position 4245. TheAatII-restriction site is incorporated at the 5′ end of the sense primerand after AatII/AgeI digestion, a 762 by fragment is generated. Bothfragments were gel-purified.

The vector pWE.Ad11.dNdeI was digested with AatII and AgeI andgel-purified. Subsequently, the AatII-AgeI fragment frompPCRScript.pIX.dPr was cloned into the AatII-AgeI-digestedpWE.Ad11.dNdeI vector resulting in pWE.Ad11.dNde.dPr (FIG. 30).Moreover, the AatII-AgeI fragment from pPCRScript.pIX.dH.dPr was alsocloned into AatII-AgeI-digested pWE.Ad11.dNdeI, which resulted inpWE.Ad11.dNdeI.dH.dPr (FIG. 31). pWE.Ad11.dNdeI.dPR andpWE.Ad11.dNdeI.dH.dPr are constructs that have the same sequences aspWE.Ad11.dNdeI but lack the pIX promoter region. The reason for takingthis out is the following. In Ad35 it seems that there is an increase intransgene stability if the Ad5 pIX promoter is used instead of theoriginal Ad35 pIX promoter (see co-pending application PCT/NL02/00281).Since Ad11 is closely related to Ad35 based on amino acid and nucleotidehomology, it was reasoned that the pIX promoter from Ad5 would also bebeneficial to the transgene if Ad11 would be used as a backbone. Forthis, also Ad11 constructs were generated harboring the pIX promoterfrom Ad5. It is not desirable to have the pIX promoter in the overlapbetween the adapter plasmid and the cosmid when homologous recombinationis applied in for instance eukaryotic cells during complementation andgeneration of the virus. Whenever one changes something in the pIXpromoter of the adapter, one would be obliged to make the same change inthe cosmid vector in order to maintain the sequence homology and toprovide proper homologous recombination. Thus, it is more efficient ifthe promoter region is not part of the overlap, and only present in theadapter plasmid (plasmids are easier to handle than cosmids). This isthe reason that the Ad11 pIX promoter is removed from the cosmid.

In pWE.Ad11.dNdeI.dPr, the original HindIII site is still present atposition 3934 located in the pIX region of Ad11. This site has beenchanged into a NheI site in pWE.Ad11.dNdeI.dH.dPr.

Both cosmid vectors pWE.Ad11.dNdeI.dPr and pWE.Ad11.dNdeI.dH.dPr weredigested with NdeI and gel-purified. DNA was isolated from wild-typeAd11 viral particles and digested with NdeI-yielding fragments of 1.63kb, 6.54 kb and 26.63 kb, respectively. The largest NdeI fragment (26.63kb) was purified from low-melting point agarose gel (1%) using agaroseenzyme (Roche) according to the manufacturer's instructions. Thisfragment was ligated into both the NdeI-digested cosmid vectors (seeabove) and packaged using the A, packaging extracts (Stratagene)according to the manufacturer's protocol. After infection into STLB-2bacteria, colonies were grown on plates and analyzed for the presence ofthe complete insert. Recombinant clones were analyzed by restrictionenzyme digestion with NheI, HindIII, ScaI, ApaLI, BamHI and HpaI. Thetwo cosmids that were generated after inserting the 26.6 kb (Ad11-NdeI)fragment in pWE.Ad11.dNdeI.dPr and pWE.Ad11.dNdeI.dH.dPr were namedpWE.Ad11 COSMID and pWE.Ad11.dH COSMID, respectively.

Example 11 Validation of a Quantitative Adenovirus Neutralization Assay,Based on Luciferase-Transgene Detection

Currently, different assays are being used to determineanti-adenovirus-neutralizing activity. As input virus either wild-typeadenovirus (wt-Ad) or recombinant replication deficient Ad5 is used.With wt-Ad, cell lines that support replication are needed. The read-outis usually performed microscopically by scoring the adenovirus-mediatedcytopathic effect (CPE) or automated by staining for cell viability. Theresults from such an “Adenovirus Replication Inhibition Assay” (ARIA)are highly dependent on the timing of read-out but usually take betweenfour to eight days. In another assay, replication deficient Ad virus isused and inhibition of transgene expression is taken as a parameter forantiviral-neutralizing activity. For such an “Adenovirus TransgeneExpression Inhibition Assay” (ATEIA), recombinant adenoviruses carryingthe LacZ, GFP or luciferase encoding genes as reporter can principallybe used. This wide range of available assays, none of which isvalidated, renders published results of different studies difficult tointerpret and compare, and thus shows a need for standardization.

Here, a head-to-head comparison of different protocols used to date todetermine anti-Ad5-neutralizing activity is described. Based onaccuracy, robustness, simplicity, and sensitivity, a neutralizationassay based on recombinant Ad5-carrying luciferase is proposed asread-out inhibition of luciferase transgene expression.

Human Sera and IgG

Serum samples were derived from healthy adult volunteers in Belgium. Thesamples were screened for antibodies present against wt-Ad as describedin Pauwels et al. (1988). Samples from 18 Ad5-seropositive donors werepooled and samples from five Ad5-seronegative were pooled. As a positivecontrol, the “National Institute for Biological Standards and Controls(NIBSC, UK) second international standard anti-measles serum, human andsecond international standard anti-poliovirus serum, types 1, 2, and 3”was used. IgG was purified from human serum pools with the use of MabTrap Kit according to the manufacturer's protocol (Amersham PharmaciaBiotech).

Cells and Viruses

A549 human lung carcinoma cells were grown in Dulbecco's modifiedEagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and1% penicillin/streptomycin. Adenoviral vectors used were wt-Ad5,Ad5.Luc, Ad35.Luc, Ad5.GFP, Ad5.LacZ. Briefly, virus produced on T175triple-layer tissue culture flasks was purified with a two-step CsClpurification protocol. After purification, virus was aliquoted andstored at −80° C. Virus titer expressed in virus particles (vp) permilliliter was determined by HPLC.

Virus Titration

For each cell-line used the infectious titer of the virus wasdetermined. A virus serial doubling dilution in medium ranging from400,000 vp/cell to 5 vp/cell, was added to 1×10⁴ cells/well in a 96-wellplate (Greiner). After an incubation of 24 hours at 37° C. and 10% CO₂,the medium was aspirated, and 100 ml PBS followed by 100 ml Steady-GloLuciferase Assay System Reagent (Promega) was added to each well. Afteran incubation of 15 minutes at room temperature, 100 ml of each well wastransferred to a Black and White isoplate (Perkin Elmer) andluminescence counts measured on the 1450 Microbeta Trilux. The amount ofvp/cells corresponding to upper values within the linear range ofluciferase activity is used for further experiments (500 vp/cell for Ad5and Ad35 on A549 cells).

Adenovirus Neutralization Assay

Sera were heat inactivated at 56° C. for 60 minutes, before a serialdoubling dilution was performed in a 96-well tissue culture plate. Thedilutions covered the range from 12.5 ml to 6 nl serum in a volume of 50μl DMEM (eventually resulting in dilutions from 1/16 to 1/32,768 in anend volume of 200 μl). No serum was added to the negative controls,which resulted in the maximum luciferase activity. This value is used tocalculate the 90% and 50% neutralization values. To every well, 50 ml ofvirus solution was added with a multiplicity of infection (MOI) that wasdetermined by the virus titration. A cell suspension was made of 1×10⁵A549 cells/ml and 100 μl was added to every well. Plates were incubatedfor 24 hours at 37° C. and 10% CO₂ before read-out.

Neutralization Assay Read-Outs

The replication rate of wild-type adenovirus (using PER.C6 cells) wasscored by measuring cell viability and by using an MTT staining, asdescribed previously (Pauwels et al. 1988). The read-out foradenoviruses carrying luciferase is described above. For experimentsusing adenoviruses carrying GFP, medium was aspirated, 100 μl PBS addedand fluorescent levels were measured in a fluorescent plate reader(Fluoroskan Ascent FL Labsystems) using wavelengths of 485 nm(excitation) and 527 nm (emission). For experiments using adenovirusescarrying LacZ, the medium was aspirated and cells were fixed with 1%formaldehyde, 0.2% glutaraldehyde in PBS for 10 minutes at roomtemperature. After washing the cells twice with PBS, the cells wereincubated at 37° C. in a 2.5 mM X-gal(5-bromo-4-chloro-3-indolyl-β-galactosidase, Invitrogen, Grand Island,N.Y.) reaction mixture containing 5 mM K₄Fe(CN)₆, 5 mM K₃Fe(CN)₆ and 2mM magnesium chloride in PBS. After 4 hours of incubation, plates weremeasured on an ELISA plate-reader (Bio-Tek Instruments Inc., Power Wave340) at 495 nm. The 90% (or 50%) inhibition serum titer is correspondingto 10% of the maximum control value (no serum), interpolated in theserum dilution range.

Quantification of Adenoviral Genomes Per Cell by Q-PCR

Total DNA was isolated from infected A549 cells with the DNeasy tissuekit (Qiagen, Germany). The Q-PCR protocol is derived from Klein et al.(1999). CMV promoter was used as target sequence, which is present inall recombinant adenoviruses used in this study. The primers and probeused in this study were CMV-F353 (5′-CAT CTA CGT ATT AGT CAT CGC TAT TACCA-3′SEQ ID NO:15), CMV-R446 (5′-TGG AAA TCC CCG TGA GTC A-3′ SEQ IDNO:16) and probe CMV-2 (5′-VIC ACC GCT ATC CAC GCC CAT TGA TGT TAMRA-3′SEQ ID NO:17). A second pair of oligonucleotides and a probe recognizing18S rDNA was added to the reaction to make determination of virusparticles per cell possible (Klein et al. 2000). As standard fordetermination of the adenoviral genomes and number of cells present, theCMV promoter containing plasmid pAdApt35IP1 and human genomic DNA wereused, respectively. Amplification was performed in an ABI Prism 7700sequence detection system (Perkin-Elmer).

To compare the ARIA and the ATEIA assays, a small panel of human serumsamples was tested for anti-Ad5 antibody titers by ARIA, and in parallelwith the ATEIA. The results, shown in FIG. 32, indicate that the ATEIAis more sensitive than the ARIA. Furthermore, neutralizing activitytiters (50% and 90%) correlated better in the ATEIA than in the ARIA.Thus, based on sensitivity, a lower amount of serum required andsignificantly shorter time needed until read-out, the ATEIA ispreferred.

One important parameter dictating the usefulness of this assay is thepossibility to use small volumes of serum for high-through-put analyses.Hereto, the detection limit was determined by testing three differenttransgenes and their corresponding read-out system in combination withlow cell numbers (10¹ to 10⁵ cells/well). For high-through-put purposes,cells infected with Ad5.LacZ were measured by optical densitymeasurement, which proved successful in that obtained results arerepresentative for the transduction inhibition as measured by countinginfected cells using a microscope. From the results obtained (FIG. 33),it could be concluded that with LacZ and GFP, neutralizing antibodiescan be detected only when 10⁴ cells/well were seeded, even when the newread-out method for Ad5.LacZ is used as described. In contrast,luciferase activity could still be detected when using 10³ cells/well.Moreover, these results showed that when more cells per well seeded, theassay becomes more sensitive.

The assay is intended to determine the inhibition of virus infection bymeasuring luciferase activity. To determine whether serum decreasedactual virus entry into target cells, and to exclude that high serumconcentrations killed target cells, thereby diminishing transgeneexpression, transgene detection (measurement of luciferase activity) wascombined with cellular adenovirus genome detection (by Q-PCR).Simultaneous detection of the number of virus copies of Ad5 and Ad35 percell and luciferase activity showed that transgene expression wascorrelated with Adenovirus genomes per cell, and that high serumconcentrations both decreased luciferase and cellular adenovirus copiesby its neutralizing activity (FIGS. 34B and 34C). Serum does notinterfere with Q-PCR results, as the positive controls with Ad35 arepositive throughout the serum dilution. These results show thatneutralization takes place mainly extra-cellularly, not after virusentry in cellular vesicles, and that the assay specifically measuresinhibition of virus infection, but not secondary effects of serum.

To validate the luciferase-based ATEIA, the assay was performedindependently for five times in duplicate to assess precision andreproducibility (FIG. 34A). Given the low standard deviations it wasconcluded that the assay is well reproducible. Inter-assay variation wascalculated by transforming the standard deviation of five repetitivemeasurements into percentages. In this experiment the serum dilutionneeded for 90% neutralization is 1260±220. In percentages this is avariation of 17% (FIG. 34D). Intra-assay variation, the standarddeviation within one assay gave a serum dilution of 1390±274 for 90%neutralization. This is a variation of 20% (FIG. 34D). These data showthat the assay is well reproducible with acceptable standard deviations.

Naturally, validation of an assay requires the presence of a standardpositive control serum, one that is sufficiently characterized andreadily available. One such standard could be the second “InternationalStandard for anti-measles and anti-poliovirus human serum, types 1, 2,and 3,” obtained from NIBSC (National Institute for Biological Standardsand Control) provided that this serum neutralizes Ad5. For this purposethe standard serum was tested and it was found that indeed it containsneutralizing antibodies against recombinant Ad5. This positive controlserum was titered with ATEIA on neutralizing activity for 1/2550 (50%)and 1/625 (90%) respectively.

To determine the robustness of the luciferase-based ATEIA, severalfactors were investigated that may influence the outcome of the assay.One factor may be the used cell line. The luciferase neutralizationassay was performed standard on A549 cells as this cell line is highlysusceptible to adenovirus infection of both Ad5 and Ad35. For severalcell-lines including 3T3 (mouse fibroblasts), C2C12 (mouse myoblasts),human and murine dendritic cells, Ad5- and Ad35-neutralizing activity ofAd5-positive serum (either human or mouse) was tested. As all cells havea different infectious titer, this was established first for bothviruses. This showed that the maximum luciferase activity varies amongdifferent cells, as it is receptor dependent. This somewhat influencesthe 90% and 50% neutralization values. However, each cell line showedthat the Ad5-positive serum neutralized Ad5 and did not neutralize Ad35(data not shown), indicating the relative flexibility of the assay inthe use of cell lines.

Furthermore the effect of the sequence of events was tested, i.e.,whether A549 cells should be attached to the wells bottom beforeexposure to serum and virus, or whether cells can be added after serumand virus are mixed. But no difference was observed (data not shown).Therefore the cells can be added after diluting serum and adding virusparticles, which is easier and faster. Moreover, it was tested if thereis an effect of the incubation time of serum and virus before cells areadded. The incubation of serum and virus was varied from 0.5 to 60minutes, but no differences in results were found.

To demonstrate that the neutralizing effect of serum is mediated throughantibodies, the assay was performed with isolated IgG. IgG isolation wasconfirmed by gel-electrophoresis and Coomassie blue staining (data notshown). FIG. 35, Panel A, shows the neutralization capacity ofAd5-positive and -negative serum from which the IgG was isolated and theneutralizing activity of the isolated IgG fractions. The Ad5-positiveserum and its isolated IgG show neutralization, whereas the Ad5-negativeserum and its isolated IgG do not show neutralization. In FIG. 35, PanelB, negative serum is spiked with IgG isolated from Ad5-positive serumand with IgG isolated from Ad5-negative serum. For 90% neutralization,1.5 μg IgG is required. This demonstrates that neutralization activitycan be transferred from a positive to a negative serum sample throughIgG antibodies.

REFERENCES

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1.-38. (canceled)
 39. A method of delivering a nucleic acid molecule ofinterest to a subject utilizing an adenoviral delivery vehicle, themethod comprising: administering to the subject a recombinant adenovirusvector of Ad35 serotype having a nucleic acid molecule encoding a humanimmunodeficiency virus (HIV) antigen; and administering to the subject,subsequent to administering the recombinant adenovirus vector of theAd35 serotype, a recombinant adenovirus vector of Ad26 serotype having anucleic acid molecule encoding essentially the same HIV antigen.
 40. Amethod of delivering a nucleic acid molecule encoding an HIV antigen toa subject, the method comprising: administering a recombinant adenovirusvector of Ad26 serotype to a subject previously administered arecombinant adenovirus vector of Ad35 serotype, wherein the recombinantadenovirus vector of Ad35 serotype and the adenovirus vector of Ad26serotype each comprise a nucleic acid molecule encoding essentially thesame HIV antigen.
 41. A kit of parts comprising a priming compositionand a boosting composition, the kit of parts comprising: a primingcomposition comprising: a first recombinant adenovirus vector of Ad35serotype comprising a nucleic acid molecule encoding an HIV antigen, anda pharmaceutically acceptable carrier; and a boosting compositioncomprising a second recombinant adenovirus vector of Ad26 serotypecomprising a nucleic acid molecule encoding an HIV antigen and apharmaceutically acceptable carrier.
 42. The kit of parts of claim 41,further comprising instructions to administer the priming compositionand the boosting composition to a subject, wherein the boostingcomposition is to be administered subsequent to the priming composition.43. A method of delivering a nucleic acid molecule encoding an HIVantigen to a subject, the method comprising: screening a subject for thepresence of neutralizing antibodies against an adenovirus of Ad35serotype; and administering to the subject a recombinant adenovirusvector of a Ad35 serotype encoding an HIV antigen, and subsequentlyadministering to the subject a recombinant adenovirus of Ad26 serotypeencoding essentially the same HIV antigen.
 44. A method of delivering anucleic acid molecule of interest to a subject utilizing an adenoviraldelivery vehicle, the method comprising: administering to the subject arecombinant adenovirus vector of Ad26 serotype having a nucleic acidmolecule encoding an HIV antigen; and administering to the subject,subsequent to administering the recombinant adenovirus vector of theAd26 serotype, a recombinant adenovirus vector of Ad35 serotype having anucleic acid molecule encoding essentially the same HIV antigen.
 45. Amethod of delivering a nucleic acid molecule encoding an HIV antigen toa subject, the method comprising: administering a recombinant adenovirusvector of Ad35 serotype to a subject previously administered arecombinant adenovirus vector of Ad26 serotype, wherein the recombinantadenovirus vector of Ad26 serotype and the adenovirus vector of Ad35serotype each comprise a nucleic acid molecule encoding essentially thesame HIV antigen.
 46. A kit of parts comprising two immunologicalcompositions, the kit of parts comprising: a first immunologicalcomposition comprising: a first recombinant adenovirus vector of Ad35serotype comprising a nucleic acid molecule encoding an HIV antigen, anda pharmaceutically acceptable carrier; and a second immunologicalcomposition comprising: a second recombinant adenovirus vector of Ad26serotype comprising a nucleic acid molecule encoding an HIV antigen, anda pharmaceutically acceptable carrier, wherein the HIV antigen in thefirst recombinant adenovirus and second recombinant adenovirus areessentially the same.
 47. The kit of parts of claim 46, furthercomprising instructions to administer the first immunologicalcomposition and the second immunological composition to a subject,wherein the second immunological composition is to be administeredsubsequent to the first immunological composition.
 48. The kit of partsof claim 46, further comprising instructions to administer the firstimmunological composition and the second immunological composition to asubject, wherein the first immunological composition is to beadministered subsequent to the second immunological composition.
 49. Themethod according to claim 39, wherein the HIV antigen is selected fromthe group consisting of gag, pol, nef, and env.
 50. The method accordingto claim 40, wherein the HIV antigen is selected from the groupconsisting of gag, pol, nef, and env.
 51. The kit of parts of claim 41,wherein the HIV antigen is selected from the group consisting of gag,pol, nef, and env.
 52. The method according to claim 43, wherein the HIVantigen is selected from the group consisting of gag, pol, nef, and env.53. The method according to claim 44, wherein the HIV antigen isselected from the group consisting of gag, pol, nef, and env.
 54. Themethod according to claim 45, wherein the HIV antigen is selected fromthe group consisting of gag, pol, nef, and env.
 55. The kit of parts ofclaim 46, wherein the HIV antigen is selected from the group consistingof gag, pol, nef, and env.
 56. The kit of parts of claim 47, wherein theHIV antigen is selected from the group consisting of gag, pol, nef, andenv.
 57. The kit of parts of claim 48, wherein the HIV antigen isselected from the group consisting of gag, pol, nef, and env.